In vivo gene therapy using intraosseous delivery of a lentiviralgene construct

ABSTRACT

Methods, compositions, and systems for treating subject(s) in need of plasma Factor VIII, particularly a subject having preexisting anti-FVIII inhibitory antibodies, are provided. The methods involve administering to the subject a therapeutically effective amount of an inflammation suppressor, a therapeutically effective amount of a CD8+ T cell depleting agent, and a therapeutically effective amount of a composition comprising a lentiviral vector (LV) comprising an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter. Such methods, compositions, and systems are useful to treat subjects with blood clotting disorder(s), such as hemophilia A.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/663,930 filed on Apr. 27, 2018, which is incorporated herein byreference in its entirety as if fully set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.HL123326-01A1 and HL134621-01, awarded by the NIH/NHLBI. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure provides compositions and methods for in vivogene therapy, for instance using intraosseous delivery or other deliverymodes. Examples of such therapy provide platelet-specific expression ofactivity-enhanced plasma Factor VIII, for instance for treatment ofhemophilia and other blood clotting disorders.

BACKGROUND OF THE DISCLOSURE

Inhibitory antibody formation against human factor VIII (FVIII) is asignificant complication in treatment of hemophilia A (HemA) patients.Ectopic expression of FVIII in platelets could be an effective approachfor treating HemA. Unlike circulating plasma FVIII, FVIII stored inα-granules of platelets is protected from being processed by antigenpresenting cells, therefore significantly reducing the chance to induceanti-FVIII immune responses. Moreover, platelet FVIII is not neutralizedby preexisting anti-FVIII inhibitory antibodies (inhibitors), which willbenefit HemA patients who already developed inhibitors. During bleeding,the activated platelets locally excrete FVIII that can directlyparticipate in the coagulation cascade and promote clot formation. Ithas recently been demonstrated that low levels of platelet FVIII canpartially correct hemophilia phenotype in animal models.

One approach to direct FVIII long-term expression in platelets isintraosseous (IO) infusion of lentiviral vectors (LVs) carrying a FVIIItransgene controlled by a megakaryocyte specific promoter Gp1bα(Gp1bα-F8-LVs). In this in vivo gene therapy protocol of HemA,hematopoietic stem cells (HSCs) was efficiently transduced by LVs insitu, followed by FVIII transgene expression in the late stage ofmegakaryocyte differentiation, and finally storage of FVIII protein inα-granules of platelets. Previous work showed that a single IO infusionof Gp1bα-F8-LV resulted in over 3% platelets containing hFVIII, leadingto partial phenotype correction in immune competent HemA mice with andwithout pre-existing inhibitors.

Furthermore, in vivo delivery of LVs can avoid many difficulties andpotential toxicities encountered by ex vivo gene therapy including lossof stem cell properties and engraftment potential after cell transfer,negative effects of ex vivo cytokine stimulation and pre-conditioning ofthe subject. However, one potential limitation of in vivo gene therapyprotocols is the induction of LV-specific immune responses, which maydecrease LV transduction efficiency and eliminate LV transduced cells.

HemA, characterized by a deficiency of functional plasma FVIII, is anideal disease candidate for gene therapy to attain long-term therapeuticFVIII levels. An optimized transgene for effective in vivo gene therapyof hemophilia would confer a higher gene expression level and atransgene product with enhanced biological activity. Since the size of afull-length human FVIII cDNA is quite large (>7 kbp), it cannot beeasily packaged in viral vectors, often resulting in low titers ofviruses. Shorter cDNAs coding for FVIII variants have therefore beenused in gene therapy preclinical research. For instance, a B-domaindeleted FVIII (BDD-FVIII) variant exhibits similar functional activityas the full-length FVIII. Additional mutations have been incorporatedinto the gene coding for the BDD-FVIII to increase its secretion, suchas, F8/N6 with a 226 aa B-domain variant sequence (Miao et al., Blood103:3412-3419, 2004), codon optimized BDD-FVIII and F8/N6 (Ward et al.,Blood 117:798-807, 2011), F8-V3 (McIntosh et al., Blood 121:3335-3344,2013) with a 17-aa peptide coding sequence replacing the 226 aa/N6 inF8/N6, and F8-RH encoding a BDD-FVIII variant with R1645H (Siner et al.,Blood 121:4396-4403, 2013). Some FVIII variants, like FVIII-RH andfurin-cleavage site deleted BDD-FVIII variants (Nguyen et al., J ThrombHaemostas. 15:110-121, 2017), exhibit an increase in biological activitycompared with BDD-FVIII, likely due to its slower dissociation of theA2-domain upon thrombin activation.

U.S. Pat. No. 5,779,708 describes an intraosseous and/or hard tissuedrug delivery device and method that perforates hard tissue and providesa drug delivery passage into underlying tissue. The device includes ahollow drill bit and a stylet removably inserted into the hollow drillbit. The stylet, which keeps the bore of the hollow drill bit fromplugging up during drilling, is removed after drilling to permit theoperator to inject medication through the bore of the hollow drill bit.

In spite of previous work, there still exists a need for safe andreliable methods of replacing or supplementing functional FVIII in asubject. There is a particular need for methods which do not rely onpre-conditioning or myeloablative treatments, and which can providetherapeutic benefit (such as appropriate clotting function) in spite ofthe presence of inhibitory anti-FVIII antibodies in the subject to betreated.

SUMMARY OF THE DISCLOSURE

Described herein is the development of clinically relevant strategiesfor treating HemA. In examples of these strategies, self-inactivatinglentiviral vectors (LV) carrying various different FVIII varianttransgenes (including variants optimized for expression in this system)driven by a platelet-specific promoter Gp1bα (G) were delivered intoHemA subjects without preconditioning. In some embodiments, thistransgene is delivered via intraosseous (IO) administration (forinstance into the tibia or iliac bone). In other embedment's, deliveryis intranasal (or otherwise to the lung) or intravenous. The providedFVIII transgenes can effectively transduce hematopoietic stem cells(HSCs), largely without elicitation of anti-FVIII inhibitory antibodies(anti-FVIII inhibitors). The resulting FVIII is expressed and stored inplatelet α-granules, and can at least partially correct bleedingphenotype in immune-competent HemA subjects, even in the presence ofpre-existing anti-FVIII inhibitors.

Embodiments of the current disclosure describe compositions and methodsthat enable combination treatments including IO delivery of LVsexpressing a platelet-targeted modified FVIII gene. for treatment ofhemophilia. The use of IO delivery for efficient transduction of HSCs invivo results in a low inflammatory and/or immune response in thesubject, accompanied by low systemic toxicity. This is particularlybeneficial for certain subjects, including for instance subjects with ableeding disorder such as hemophilia.

Additional embodiments provide a method of treating a subject in need ofplasma Factor VIII, including: administering to the subject atherapeutically effective amount of an inflammation suppressor (such asdexamethasone (Dex)); administering to the subject a therapeuticallyeffective amount of a CD8+ T cell depleting agent; and administering tothe subject a therapeutically effective amount of a compositionincluding a lentiviral vector including an optimized FVIII expressioncassette expressibly linked to a megakaryocyte-specific promoter,wherein administration is via intraosseous (IO) infusion; intravenous(IV) delivery; or intranasal (IN) or inhaled delivery.

In examples of this method of treatment embodiment, the dexamethasone isadministered in doses of 100 mg/kg at −24 h before, −4 h before, 4 hafter, and 24 h after administration of the LV.

In examples of the provided embodiments, the CD8+ T cell depleting agentincludes an anti-CD8 antibody. For instance, in examples, the CD8+ Tcell depleting agent is an anti-CD8α mAb administered in doses of 4mg/kg at −1 day before, 4 days after, and 11 days after delivery of theLV.

In other examples of the provided embodiments, themegakaryocyte-specific promoter is a GP1b-alpha promoter.

Specifically contemplated in examples of the provided embodiments aremethods that do not include pre-conditioning or myeloablative treatmentof the subject.

In more examples of the provided embodiments, the IO infusion is carriedout at a rate of 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at nomore than 10 μL/min. For instance, the IO infusion in some examples iscarried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.Optionally, the IO infusion is carried out over a period of no more than45 minutes.

In yet additional examples, the optimized FVIII expression cassetteincludes hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH (SEQ ID NO: 2). In anyof the provided embodiments, the lentiviral vector may include SEQ IDNO: 3 or SEQ ID NO: 4, or a functional variant thereof.

Also provided are examples of the herein provided methods, which aremethods for treating a hemostasis related disorder in the subject, andthe subject is in need of such treatment. By way of example, the subjectmay have hemophilia A (hemA), von Willebrand disease, bleedingassociated with trauma or injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated vascular coagulation (DIC), orover-anticoagulation treatment disorder.

In yet another embodiment of the provided treatment method, the subjectis a HemA subject with preexisting anti-FVIII inhibitory antibodies.

Also provided are methods of treating a subject in need of plasma FactorVIII, essentially as described herein.

BRIEF DESCRIPTION OF THE FIGURES

At least one of the drawings submitted herewith is better understood incolor. Applicant considers the color versions of the drawing(s) as partof the original submission and reserve the right to present color imagesof the drawings in later proceedings.

FIGS. 1A-1B: GFP expression in bone marrow cells following IO infusionof M-GFP-LV in immune-competent and immune-deficient mice. Both C57BL/6J(BL6) and B6(Cg)-Rag2^(tm1.1Cgn)/J (Rag2^(−/−)) mice were intraosseouslyinfused with self-inactivating LV encoding GFP under the control of themodified myeloid proliferative sarcoma virus promoter (MND) (M-GFP-LV)(1.1×10⁸ ifu/animal) or sterile PBS (20 μl/animal, mock) on day 0. Theexperimental mice were sacrificed on day 7, day 28 and day 84. Then thebone marrow cells were isolated and GFP expression levels in total bonemarrow cells (left panels in FIGS. 1A and 1B) and bone marrowhematopoietic stem cells (HSCs, Lin⁻C-Kit⁺Sca1⁺) (right panels in a andb) were detected by flow cytometry. The data were shown asrepresentative flow images (FIG. 1A) and summary plot over time (FIG.1B). Each symbol represented an individual animal. Data were expressedas mean±SED. Differences were considered significant at p<0.01(**) andp<0.001(***). Data shown were from two independent experiments.

FIGS. 2A-2E: Transient immune suppression to enhance in situtransduction efficiency of bone marrow cells following IO infusion ofM-GFP-LVs. (FIG. 2A) Schematic of IO infusion of M-GFP-LVs into BL6 micepretreated with intraperitoneal injection of Dexamethasone (Dex, 100mg/kg, 4×, −24 h, −4 h, 4 h and 24 h) or anti-CD8α mAb (4 mg/kg, 3×, day−1, 4 and 11, or 5×, day −1, 4, 11, 16 and 21), or combined drugs (Dex4×+Anti-CD8α mAb 5×). GFP expression in total bone marrow cells and HSCs(Lin⁻C-Kit⁺Sca1⁺) was measured by flow cytometry. (FIG. 2B) BL6 micewere pretreated with Dex following IO infusion of MND-GFP-LVs (8.8×10⁸ifu/animal) or sterile PBS (20 μl/animal, mock). GFP expression in totalbone marrow cells (left panel) and HSCs (right panel) was detected onDay 7. (FIG. 2C) BL6 mice were pretreated with anti-CD8α mAb (3×)following with IO infusion of GFP-LVs (8.8×10⁷ ifu/animal) or sterilePBS (20 μl/animal, mock). GFP expression in total bone marrow cells(left panel) and HSCs (right panel) was detected on Day 9, 30 and 63.(FIG. 2D) BL6 mice were pretreated with combined drugs following with IOinfusion of GFP-LVs (3.6×10⁸ TU/animal) or sterile PBS (20 μl/animal,mock). GFP expression in total bone marrow cells (left panel) and HSCs(right panel) was detected on Day 7, 69 and 160. (FIG. 2E) Genomic DNAof the peripheral white blood cells (left panel) or of bone marrow cells(right panel) were isolated from the treated mice on day 160 in (FIG.2D). GFP-LV copy number in these blood cells were detected by real timeqPCR. Data were expressed as mean±SED. Differences were consideredsignificant at p<0.05(*), p<0.01(**) and p<0.001(***). Data shown werefrom two independent experiments.

FIGS. 3A-3C: Transient immune suppression to enhance phenotypecorrection in hemophilia A mice following IO infusion of G-F8/N6-LVs.HemA mice were pretreated with combined drugs (Dex 4×+Anti-CD8α mAb 5×)and then given IO infusion of self-inactivating LVs encoding hFVIIIvariant with the proximal 226 amino acid region of the B-domain (F8/N6)under the control of Gp1bα promoter (G-F8/N6-LV, 2.2×10⁶ ifu/animal) onday 0. (FIG. 3A) HemA phenotype correction of G-F8/N6-LV orG-F8/N6-LV+drugs treated mice was evaluated by tail clip assay on day 70(n=6-8/group). The average blood loss of untreated HemA mice was set as100%. Wild-type C57BL/6 mice were used as positive controls. (FIG. 3B)HemA phenotype correction of G-F8/N6-LV+drugs or G-F8/N6-LV treated micewas also evaluated by measuring carotid artery blood flow rate on day84. (FIG. 3C) Plasma samples were collected from the G-F8/N6-LV+drugs orG-F8/N6-LV treated mice on day 84. hFVIII activity and anti-FVIIIantibodies were measured by aPTT and Bethesda assay, respectively. Thelevel of hFVIII activity in untreated HemA mice was corrected as 0% ofnormal and its level of anti-FVIII antibodies was 0 Bethesda unit. Eachsymbol represented an individual animal. Data were expressed asmean±SED. Differences were considered significant at p<0.01(**),p<0.001(***) and p<0.0001(****).

FIGS. 4A-4B: BDDF8X10K12 Generated Higher Expression Levels. (FIG. 4A)Schematic of a new human FVIII cDNA variant. Compared to F8/N6, F8X10K12had a deleted B-domain, a 10-amino acid change in the A1 domain and a12-amino acid change in the light chain. (FIG. 4B) Both BDDF8X10K12 andF8/N6 were cloned into a lentiviral transgene backbone plasmidcontrolled under a ubiquitous promoter EF1α (pEF1α-F8X10K12 andpEF1α-F8/N6), respectively. Then HemA mice were hydrodynamicallyinjected with pEF1α-F8X10K12 (n=3) or pEF1α-F8/N6 (n=9) (50 μg/animal)or sterile PBS (mock, 2 ml/animal). Plasma samples were collected on day4 post injection and hFVIII activity was measured by aPTT. Data wereexpressed as mean±SED. Differences were considered significant atp<0.0001 (****). (FIG. 4C) Both E-F8X10K12-LV and E-F8/N6-LV weregenerated to transduce 293T cells (MOI=100) on day 0. On day 5, hFVIIIexpression levels in 293T cells were detected by flow cytometry.

FIGS. 5A-5F: Phenotype correction was achieved in drugs pretreated HemAmice following with IO infusion of G-F8X10K12-LVs. (FIG. 5A) Schematicof a self-inactivating LV genome encoding F8X10K12 under the control ofplatelet-specific glycoprotein 1bα promoter (Gp1bα) (G-F8X10K12-LV).(FIG. 5B) HemA mice were pretreated with drugs (Dex 4×+Anti-CD8α mAb 5×)following with IO infusion of G-F8X10K12-LVs (2.2×10⁶ ifu/animal), orwere only given IO infusion of G-F8/N6-LV (2.2×10⁶ ifu/animal) orG-F8X10K12-LVs (2.2×10⁶ ifu/animal) or sterile PBS (mock, 20 μl/animal)on day 0. hFVIII levels in platelet lysates in G-F8X10K12-LV+drugstreated mice or G-F8X10K12-LV only treated or G-F8/N6-LV only treated ormock were measured by ELISA on day 90. Each symbol represented anindividual animal. (FIG. 5C) Anti-FVIII antibodies in the plasma samplescollected from the treated and mock mice were measured by Bethesda assayon day 120. The level of anti-FVIII antibodies in mock mice was 0Bethesda unit. Each symbol represented an individual animal. (FIG. 5D)LV copy numbers in peripheral white blood cells of treated or mock weredetected by qPCR on day 120. Each symbol represented an individualanimal. (FIG. 5E) HemA phenotype correction the treated mice wasmonitored by tail clip assay on day 120. The average blood loss ofuntreated HemA mice was set as 100%. Wild-type C57BL/6 mice were used aspositive controls. Each symbol represented an individual animal. (FIG.5F) Platelet-rich plasma was isolated from the treated mice on day 200and functional activity of FVIII was evaluated by thrombin generationassay. The generated thrombin in the assay was evaluated by threeparameters: lag phase time, peak thrombin concentration and totalthrombin. Data were expressed as mean±SED. Differences were consideredsignificant at p<0.01(**), p<0.001(***) and p<0.0001(****).

FIGS. 6A-6D: Phenotype correction was achieved in transiently immunesuppressed HemA mice following with IO infusion of G-F8/N6K12RH-LVs.(FIG. 6A) Schematic of a new human FVIII cDNA variant—F8/N6K12RH.Compared to F8/N6, F8/N6K12RH had a 12-amino acid change in the lightchain and an amino acid change at the furin cleavage site within the Bdomain (position R1645H). F8/N6K12RH was cloned into a lentiviraltransgene backbone plasmid controlled under a ubiquitous promoter EF1α(pEF1α-F8/N6K12 RH). Then HemA mice were hydrodynamically injected withpEF1α-F8/N6K12RH (n=3) (50 μg/animal) or pEF1α-F8/N6 (n=5) (50μg/animal) or sterile PBS (n=3) (mock, 2 ml/animal). Plasma samples werecollected on day 4 post injection and hFVIII activity was measured byaPTT. (FIG. 6B) Schematic of a self-inactivating LV genome encodingF8/N6K12RH under the control of platelet-specific glycoprotein 1bαpromoter (Gp1bα) (G-F8/N6K12RH-LV). (FIG. 6C) HemA mice were pretreatedwith drugs (Dex 4×+Anti-CD8α mAb 5×) following with IO infusion ofG-F8/N6K12RH-LV (2.2×10⁶ ifu/animal) or of G-F8/N6-LV (2.2×10⁶ifu/animal) or sterile PBS (mock, 20 μl/animal) on day 0. Plasma sampleswere collected from the treated mice or mock on day 84. hFVIII activityand anti-FVIII antibodies were measured by aPTT and Bethesda assay,respectively. The level of hFVIII activity in untreated HemA mice wascorrected as 0% of normal and its level of anti-FVIII antibodies was 0Bethesda unit. (FIG. 6D) HemA phenotype correction of G-F8/N6-LV only orG-F8/N6K12RH-LV+drugs treated mice was also evaluated by measuringcarotid artery blood flow rate on day 84. Each symbol represented anindividual animal. Data were expressed as mean±SED. Differences wereconsidered significant at p<0.05(*).

FIGS. 7A-7B are schematics of hFVIII variants and LVs incorporatingcDNAs encoding GFP or hFVIII variants. FIG. 7A hFVIII variants. Comparedto F8/N6, F8X10K12 had a deleted B-domain, a 10-amino acid change in theA1 domain and a 12-amino acid change in the light chain, and F8/N6K12RHhad a 12-amino acid change in the light chain and an amino acid changeat the furin cleavage site within the B domain (position R1645H). FIG.7B Schematics of self-inactivating LV constructs encoding GFP under thecontrol of a MND promoter or various hFVIII variants including F8/N6,F8X10K12 and F8/N6K12RH under the control of a ubiquitous EF1α promoter,or platelet-specific GP1BA promoter.

FIGS. 8A-8B. Characterization of CD8α⁺CD3ε⁺ cells in blood and bonemarrow of BL6 mice after anti-CD8α monoclonal antibody treatment. (FIG.8A) Intraperitoneal injection of anti-CD8α mAb (4 mg/kg, n=3) or sterilePBS (200 μl/animal, n=3) on day 0. On day 7, blood cells were collectedand the percentage of CD8α+CD3ε+ was detected by flow cytometry. (FIG.8B) BL6 mice were pretreated with anti-CD8α mAb (4 mg/kg, 3×, day −1, 4and 11) following with IO infusion of MND-GFP-LVs (1.8×10⁸ ifu/animal,n=10) or sterile PBS (20 μl/animal, mock, n=4). Blood samples werecollected by retro-orbital bleeding and bone marrow cells were obtainedafter the treated mice or mock were sacrificed at different time points.The change of the percentage of CD8α+CD3ε over time was monitored byflow cytometry. Data were expressed as mean.

FIGS. 9A-9B. Representative flow images of GFP expression in total bonemarrow cells (FIG. 9A) and bone marrow hematopoietic stem cells (HSCs,Lin⁻C-Kit⁺Sca1⁺) (FIG. 9B) in FIG. 2C.

FIGS. 10A-10B. Representative flow images of GFP expression in totalbone marrow cells (FIG. 10A) and bone marrow hematopoietic stem cells(HSCs, Lin⁻C-Kit⁺Sca1⁺) (FIG. 10B) in FIG. 2D.

FIGS. 11A-11B illustrate proof-of-principle research models in humanizedNSG mice. FIG. 11A is a flow chart showing steps of methods described inExample 3. FIG. 11B is schematic drawings of two lentiviral (LV)constructs used in the studies described in Example.

FIGS. 12A-12B. Human CD34+ cells were cultured in SFEM II supplementedwith cytokine cocktail for expansion of human hematopoietic cells(CC110), or serum-free culture supplement for expansion of humanmegakaryocytes (Meg) for 7 and 13 days (FIG. 12A). Human CD34+ cellscultured in SFEM II+Meg were transduced with M-GFP-LV or G-GFP-LV (FIG.12B). Megkaryocytes and GFP expression on day 7 were detected by flowcytometry.

FIGS. 13A-13C. 6-week NSG mice were retro-orbitally injected with 1×10⁶human CD34+ cells one day after preconditioning with busulphan (25mg/kg). After 13 weeks, bone marrow (FIG. 13A), spleen (FIG. 13B), andblood (FIG. 13C) were collected. Human CD34+ cells in bone marrow, andhuman CD45+ cells and murine CD45+ cells in bone marrow, spleen andblood were detected by flow cytometry.

FIGS. 14A-14B. M-GFP-LV was intraosseously infused into humanized NSGmice, which were generated by retro-orbital injection of human CD34+cells 9 weeks ago. Four weeks later, the mice were sacrificed and bonemarrow and spleen were isolated. GFP expression in total cells, humanCD45+ and murine CD45+ cells in bone marrow (FIG. 14A) and spleen (FIG.14B) was detected by flow cytometry.

FIG. 15. FVIII gene expression in canine platelets was evaluated byELISA following IO gene therapy.

FIG. 16A-16B. HemA phenotype correction was examined by (FIG. 16A) WBCTand (FIG. 16B) TEG in M80 after IO Injection.

FIGS. 17A-17H. GFP expression in BM cells following IV delivery ofMND-GFP-LVs. (FIG. 17A) The mobilization drug is used to mobilize HSCfrom BM to blood in mice. Representative flow cytometry plot of LSKs(Lineage—, sca1+, c-kit+) in peripheral blood after mobilization by GSFand AMD3100. (FIG. 17B) After mobilization, IV delivery of GFPLenti-vectors driven by a ubiquitous MND promoter was injected via retroorbital injection. The total GFP expression in PBMCs was detected atvarious time points. (FIG. 17C) LSK cells from mobilization treatmentgroup or non-mobilization group were analyzed by flow cytometry. (FIGS.17D-17F) Percentage of GFP marked cells, as determined by flow cytometerdetection in T, B and Myeloid cells after treatment day 4 to 10 weeks.(FIG. 17G) MFI was analyzed after MND-GFP-LVs transduction in total bonemarrow cells in different groups (FIG. 17H) MFI was determined afterMND-GFP-LVs transduction in HSCs of bone marrow in different groups.Mock n=3, Mobilization group n=5, non-mobilization group n=5, p<0.05(*).

FIGS. 18A-18B. Gp1bα promoter drives GFP expression in platelets afterG-GFP-LVs injection in mobilized mice. (FIG. 18A) G-GFP positiveplatelets are detected in total platelets post G-GFP-LV injection byflow cytometry. (FIG. 18B, parts 1 and 2) The flow cytometry schematicpresentation of GFP+ platelets at 2 weeks and 10 weeks. Mock n=3,Mobilization group n=5, non-mobilization group n=5. p<0.01 (**).

FIGS. 19A-19B. Circulatory FVIII in plasma of HemA mice postmobilization treatment and E-FVIII-LV delivery via IV injection. (FIG.19A) Quantity of the circulatory FVIII in plasma was examined by ELISAassay. (FIG. 19B) Activity of the circulatory FVIII in plasma wasdetected by APTT assay. Meanwhile inhibition of the FVIII in plasma wasdetected by Bethesda assay. Mobilization group n=3, non-mobilizationgroup n=3.

FIGS. 20A-20E. FVIII protein can be stored in platelets after G-FVIII-LVtreatment. (FIG. 20A) The representative flow cytometry schematic ofFVIII+ platelets at day 30 and 125. (FIG. 20B) The hFVIII intracellularstaining in platelet were measured by flow cytometer on day 12, 30,65,90 and 125. (FIG. 20C) The hFVIII levels in platelet lysates of themobilization, and the non-mobilization mice were measured by ELISA onday 125. (FIG. 20D) Copy number was detected in BM after 6 monthstreatment by TaqMan q-PCR. (FIG. 20E) FVIII inhibition in plasma wasdetected by Bethesda assay after 6 months treatment. Mock n=3,Mobilization group n=5, non-mobilization group n=5.

FIGS. 21A-21E. G-FVIII-LVs gene therapy improved the function of bloodclotting in mobilization HemA mice model. (FIGS. 21A-21D) The clottingtime (CT), clotting formation time (CFT), max clotting firmness (MCF)and a angle was detected by ROTEM® assay in wide type, HemA,mobilization and non-mobilization group. (FIG. 21E) The representativeschematic of the clotting in mobilization group (right) andnon-mobilization group (left).

FIGS. 22A-22E. Intravenous delivery of lentiviral vectors to treathemophilia A in a HSCs mobilization mouse model. (FIG. 22A) Schematicsof self-inactivating LV constructs encoding GFP under the control of aMND promoter or a platelet-specific GP1BA promoter and encodinghFVIII/N6 under the control of a ubiquitous EF1α promoter, orplatelet-specific GP1BA promoter. (FIG. 22B) Schematics of the method ofmobilization of HSCs and subsequent intravenous infusion of LVs inhemophilia A mice. (FIG. 22C) Experimental schedule of mobilization ofHSCs and subsequent intravenous infusion of LVs in hemophilia A mice.(FIG. 22D) GFP expression in mice treated with mobilization andM-GFP-LVs. Mock: untreated mice, mobilized: mice treated withmobilization and LVs, non-mobilized: mice treated with LVs only. (FIG.22E) ROTEM assay results for different groups of mice, including: Panel1-5: hemophilia A mice treated with HSCs mobilization of HSCs andintravenous infusion of LVs; Panel: 6-12: hemophilia A mice treated withintravenous infusion of LVs only; Panel 13-15: hemophilia A mice; andPanel 16-18: normal wild-type mice.

FIGS. 23A, 23B. (FIG. 23A) Pictorial overview of scheme being exploredthrough treatment of Hemophilia A via intranasal (IN) delivery oflentiviral vectors encoding Factor VIII, as described in Example 8.(FIG. 23B) Illustration of vectors used in Example 8.

FIGS. 24A, 24B G-GFP expression in platelets by G-GFP-LVs transductionvia intranasal delivery is illustrated in. Mice were administrated with24 μl G-FVIII-LVs (1×10⁹ IFU/ml)/mouse/day via IN for 3 days. (FIG. 24A)The flow cytometry schematic of GFP+platelets. (FIG. 24B) GFP expressionin platelets was measured by flow cytometer after lentivirus treatment(N=4).

FIGS. 25A-25C Expression of FVIII in platelets by G-FVIII-LV viaintranasal delivery. (FIG. 25A-25B) FVIII expression was detected byintracellular staining and ELISA assay in platelets after IN treatment(N=4). (FIG. 25C) The representative schematic of the whole bloodclotting by ROTEM in HemA, wide type, and LV-treated mice at 30 daysafter IN dosing.

FIGS. 26A-26E Lung HSCs transduction by M-GFP-LVs, illustrated withControl Mouse Panels FIG. 26A (Specimen 001_Ing control_0003.fcsLymphocytes 7.19ES; Comp-Pacific Blue-A, SSC-A subset 52.4), FIG. 26B(Specimen—1_lung control_003.fcs Comp-Pacific Blue-A, SSC-A subset377146; Com-PerCP-Cy5-5-A, SSC-A subset 45.5), FIG. 26C (Specimen001_lung control_003.fcs Comp-PerCP-Cy5-5-a, SSC-A subset 171102;Comp-PE-CY7-A, Comp-APC-A subset 1.06), and FIG. 26D (Comp-PE-Cy7-A,Comp-GFP-A subset: mean: comp-GFP-A:10: Specimen 001_lungcontrol_003.fcs Comp-PE-Cy7-A, Comp-APC-A subset 1811; Comp-PE-CY7-A,Comp-TFP-A subset 0.44); and Intranasal Delivery D7 Panels FIG. 26E(Specimen 001_lung MGFP_004.fcs Lymphocytes 9.01ES, Comp-Pacific Blue-A,SSC-A subset 63.8), FIG. 26F (Specimen_001_lungMgfp_004.fcs Com-PacificBlue-A, SSC-A subset 5.75ES; Comp-PerCP-Cy5-5-A, SSC-A subset 60.8),FIG. 26G (Specimen 001_lung mgfp_004.fcs Comp-PerCP-Cy5-5-a, SSC-Asubset 349853, Comp-PE-Cy7-A, Comp-APC-A subset 1.06), and FIG. 26E(Specimen 001_lung mefp_004.fcs Comp-PE-Cy7-A, Comp-APC-A subset 3691,Comp-PE-Cy7-A, Comp-GFP-A subset 4.69). The X-axis scale on each panelis −10³, 0, 10³, 10⁴, 10⁵; the Y-axis scale on each panel is −10³, 0,10³, 10⁴, 10⁵.

SEQUENCE LISTING

The disclosed nucleic and/or amino acid sequences are shown usingstandard letter abbreviations for nucleotide bases, and one or threeletter code for amino acids, as defined in 37 C.F.R. 1.822. Only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. A computer readable text file, entitled “Sequence Listing.txt”created on or about Apr. 26, 2019, with a file size of 48 KB, containsthe sequence listing for this application and is hereby incorporated byreference in its entirety. In the accompanying Sequence Listing and/oras provided herein:

SEQ ID NO: 1 is the nucleic acid sequence of the insert BDDFVII1X10K12,which encodes the human FVIII variant F8X10K12; it differs from variantF8/N6 by a deleted B-domain, a 10-amino acid change in the A1 domain anda 12-amino acid change in the light chain.

SEQ ID NO: 2 is the nucleic acid sequence of the insert BDDFVIIIn6K12RH,which encodes the human FVIII variant F8/N6K12RH; it differs fromvariant F8/N6 at a 12-amino acid change in the light chain and an aminoacid change at the furin cleavage site within the B domain (positionR1645H).

SEQ ID NO: 3 is the nucleic acid sequence ofpRRL-GP1balpha-hF8X10K12-WPRE; the F8 cDNA insert hF8X10K12 (SEQ IDNO: 1) is at positions 2320-6738 of this sequence, which also includesthe lentiviral vector backbone, platelet-specific GP1b alpha promoter,and WPRE element inserted in the vector to make stable mRNA.

SEQ ID NO: 4 is the nucleic acid sequence ofpRRL-GP1balpha-hF8/N6K12RH-WPRE; the F8 cDNA insert hF8/N6K12RH (SEQ IDNO: 2) is at positions 2308-7414 of this sequence which also includesthe lentiviral vector backbone, platelet-specific GP1b alpha promoter,and WPRE element inserted in the vector to make stable mRNA.

SEQ ID NOs: 5 and 6 are, respectively, the nucleotide sequences offorward and reverse amplification primer for GAG.

SEQ ID NO: 7 is the nucleotide sequence of a probe for GAG.

SEQ ID NOs: 8 and 9 are, respectively, the nucleotide sequences offorward and reverse amplification primer for human Factor VIII.

SEQ ID NOs: 10 and 11 are, respectively, the nucleotide sequences offorward and reverse amplification primer for Rpl19.

DETAILED DESCRIPTION

Hemophilia A (HemA) is a X-linked inherited or acquired genetic diseasewith defective plasma factor VIII (FVIII), resulting in unstable bloodclot formation in HemA patients when internal and/or external bleedingoccurs. Described herein are gene therapy protocols to treat hemophiliaA and other bleeding disorders by direct infusion of lentiviral vectors(LVs) (or other expression units) carrying an engineered FVIII gene intobone marrow to efficiently transduce hematopoietic stem cells (HSCs).

In example embodiments, FVIII expression is targeted specifically toplatelets using a megakaryocyte-specific promoter to drive transgeneexpression; this results in FVIII-contain platelets circulating in theblood. Expression the platelets prevents or significantly inhibits FVIIIsecretion into blood, and thus prevents an anti-FVIII immune response inthe subjects so treated. Immune response to FVIII disadvantageouslyleads to neutralization of this factor by anti-FVIII antibodies (socalled anti-FVIII inhibitors) in hemophilia patients. Expression insideof platelets maintains the FVIII inside the platelets until they aretriggered by the clotting cascade, leading to release of FVIII whenbleeding occurs.

Based on results reported here, it is believed that a single genetherapy treatment via in vivo delivery of G-F8-LVs driven by amegakaryocyte-specific promoter directly into bone marrow will benefitHemA patients, especially patents with anti-FVIII inhibitors.

Beneficially, and in contrast to traditional ex vivo gene therapy, theIO in vivo delivery system provided herein avoids the requirement forboth ex vivo cell manipulation as well as use of fully or partiallymyeloablative condition regimens. Such pre-conditioning is usuallyrequired for successful ex vivo gene therapy.

In embodiments described herein, it can be beneficial to preciselycontrol the speed (rate) of IO infusion, as this influences theefficiency at which the IO delivered LVs are transduced into cells inthe bone marrow (such as HSCs). Low delivery speeds can be used, and maybe beneficial in smaller subjects. Relatively low speeds include, butare not limited to 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or atno more than 10 μL/min. One particular relatively low speed is 0.01mL/min. A relatively higher delivery speed (rate) can be used in largeranimals, including humans. By way of example, a relatively higher rateof delivery is carried out at 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.Clearly, a faster rate of delivery will shorten the infusion time, andis therefore considered beneficial so long as it is selected to be sucha rate that will still achieve efficient transduction in the specificsubject being treated. By way of example, he total volume of the vectorbolus used to treat a relatively large subject (such as a dog) may be2-20 mL or more, and it may be beneficial to the subject to completeinfusion in 20-40 mins.

Compared to existing, proposed treatments for hemophilia A or B thatrely on recombinant adeno-associated viral (AAV) vectors (rAAV),lentiviral gene therapy as described herein has several advantages.First, AAV persists as episomal, concatemerized vector following in vivogene transfer. Over time in that situation, transgene expression maydecrease and it is expected that repeated dosing will be required. Itremains to be shown whether such repeated dosing is feasible, or effect.In contrast, lentiviral vectors are an integrative vector that isincorporated into the genome of the transduced cells. If successfullyintegrated into long-lived bone marrow HSCs, as taught here, a singletreatment of LV-mediated gene therapy may be sufficient for life-longtherapeutic benefit. In addition, a significant proportion of humansubjects have pre-existing neutralizing antibodies to various AAVserotypes, and nearly all subjects will develop antibodies to theserotype utilized for initial treatment. This will make repeated dosingwith the same serotype challenging if not impossible.

AAV is also a poor choice for expression of FVIII cDNA because itstransgene capacity is simply too small. Further, LV can efficientlytransduce both dividing and no-dividing cells, leading to significantincreases in efficiency in targeting primitive HSCs, compared to AAV andtraditional gamma-retroviral vectors which can only transduce dividingcells.

Furthermore, recent clinical trials of ex vivo lentiviral gene therapyfor several primary deficiency diseases showed significant clinicalbenefit without adverse effects or aberrant clonal expansion.

Compared to other in vivo LV-mediated gene therapy approaches, IOdelivery is unique. Among other benefits (including benefitsspecifically described elsewhere herein), slow injection of LVs into thebone marrow cavity can avoid (substantially or completely) theinflammation and systemic toxicity caused by systemic intravenous (IV)deliver of AAV and other candidate gene therapy vectors.

In certain embodiments, it can be beneficial to use one or morecompounds to facilitate or enhance efficient transduction of the targetHSCs. For example, several compounds (such as dexamethasone, anti-CD8monoclonal antibodies, and staurosporine) can enhance LV transduction ofHSCs. Another such compound is rapamycin, and therefore example methodsinvolve the co-administration, for instance via IO, of LV and rapamycinto enhance the rate of transduction. This can be particularly beneficialin instances where the viral titer may be low or below a desired level.Another method to facilitate or enhance LV transduction efficiency is toconjugate the LVs with ligand(s) or antibody fragment(s) that canspecially recognize receptors on HSCs, thus targeting the LV to thepreferred cell for transduction.

Additional options and embodiments of the disclosure are now describedin more detail.

Any composition formulation disclosed herein can advantageously includeany other pharmaceutically acceptable carriers which include those thatdo not produce significantly adverse, allergic, or other untowardreactions that outweigh the benefit of administration, whether forresearch, prophylactic and/or therapeutic treatments. Exemplarypharmaceutically acceptable carriers and formulations are disclosed inRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990. Moreover, formulations can be prepared to meet sterility,pyrogenicity, general safety and purity standards as required by UnitedStates FDA Office of Biological Standards and/or other relevant foreignregulatory agencies.

Exemplary generally used pharmaceutically acceptable carriers includeany and all bulking agents or fillers, solvents or co-solvents,dispersion media, coatings, surfactants, antioxidants (e.g., ascorbicacid, methionine, vitamin E), preservatives, isotonic agents, absorptiondelaying agents, salts, stabilizers, buffering agents, chelating agents(e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.

Exemplary buffering agents include citrate buffers, succinate buffers,tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers,lactate buffers, acetate buffers, phosphate buffers, histidine buffersand/or trimethylamine salts.

Exemplary preservatives include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides, hexamethonium chloride, alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and3-pentanol.

Exemplary isotonic agents include polyhydric sugar alcohols includingtrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol, or mannitol.

Exemplary stabilizers include organic sugars, polyhydric sugar alcohols,polyethylene glycol; sulfur-containing reducing agents, amino acids, lowmolecular weight polypeptides, proteins, immunoglobulins, hydrophilicpolymers, or polysaccharides.

Intranasal administration is a non-invasive route for gene therapydelivery that offers advantages such as ease of administration, rapidonset of action, and avoidance of first pass metabolism, gastric acidand enzymatic degradation (Grassin-Delyle et al, Pharmacol Ther.134:366-379, 2012). Lentiviral vectors can readily be administeredintranasally. The GP64-FIV lentiviral vector has been successfullydelivered through repeated intranasal administration without significantdecline in transgene expression (Podolska et al., Adv Clin Exp Med.21:525-34, 2012). Methods of intranasal delivery include nasaldeposition or instillation, direct instillation into the trachea orlower airways using a catheter or bronchoscope, or aerosolization andinhalation (Weiss, Molecular Therapy 6:148-152, 2002).

In some embodiments, the lentiviral vector is administered intranasallyvia nasal deposition, a process wherein the site of deposition mayinfluence the route of absorption and target organ distribution. Forexample, absorption at the mucosa beyond the nasal valve is more likelyto happen through the carotid artery providing a direct route to thebrain (Djupesland, Drug Deliv Transl Res. 3:42-62, 2013).

In other embodiments, the lentiviral vector is delivered through directinstillation into the trachea or lower airways, for instance using acatheter or bronchoscope. Direct vector instillation can be performedthough bronchoscopy administration resulting in heterogenous depositionin parts of the lung, and the use of spray devices directed through thebronchoscope can improve vector distribution and deposition (Weiss,Molecular Therapy 6:148-152, 2002).

In some embodiments the lentiviral vector is delivered throughaerosolization and inhalation, a clinically convenient approach that canresult in a diffused transgene distribution in the lungs. Duringaerosolization and inhalation, different nebulization equipment andmethods can be used to produce homogenously sized aerosol dropletparticles that are deposited in bronchiolar and distal airways (Weiss,Molecular Therapy 6:148-152, 2002).

In some embodiments, the lentiviral vector is delivered as an aerosolspray from a pressure container or dispenser which contains a suitablepropellant (such as carbon dioxide), or a nebulizer (e.g., US2005/0251872), or by other methods including vapor inhalers, Rhinylecatheters, multi-dose droppers, unit-dose pipettes, squeeze bottles,multi-dose metered-dose spray pumps, single/duo-dose spray pumps, slowspray HFA pMDI's, pulsation membrane nebulizers, powder spray devicesand nasal inhalers and insufflators (Djupesland, Drug Deliv Transl Res.3:42-62, 2013).

Lentiviral vectors that carry transgenes also can be deliveredintravenously to treat human diseases. See, for instance, US2000/70190030, US 2005/0251872, CA 2,296,319, US 2007/0190030; Carbonaroet al., Mol Ther. 13:1110-1120, 2006. Intravenous (IV) administrationrefers to the process of administering a compound directly into apatient's or animal's vein using a needle or tube. Intravenousadministration is used wherein a rapid onset of action is required orwhen other delivery routes are not available due to characteristics ofthe compound or patient factors (Jin et al., Patient Prefer Adherence.9:923-942, 2015). Thus, upon injection, the compound or composition canbe rapidly delivered to organs bypassing absorptions barriers, but withthe potential to elicit toxic effects (Maddison et al., Small AnimalClinical Pharmacology, Second Edition, Chapter 2, 27-40, 2008).

Intravenous administration offers several advantages, including minimaldelay for drug availability, the possibility to achieve constant plasmaconcentrations at a required level, the avoidance of unexpected sideeffects by stopping the infusion, an alternative route when compoundsare poorly absorbed by the gastrointestinal tract, and the avoidance ofpain when other administration routes are painful (Claassen, Neglectedfactors in pharmacology and neuroscience research, Chapter 2, pp 5-22;Huston Ed., 1994).

Most intravenous injections are performed in one of the superficialveins normally used to collect blood, but the choice of vein forintravenous injection depends on several factors (well known to those ofskill in the art), including ease of venipuncture, age, whetheranesthesia or chronic catheterization is required, and type ofcomposition injected. In some embodiments, the injection of thecomposition containing a lentiviral vector is made into the jugular,caudal, femoral, lateral marginal, dorsal metatarsal, saphenous,lingual, or dorsal penile vein.

In some embodiments, the technique used to deliver a lentiviral vectorcan include a clean technique using a sterile syringe and needle, astrict aseptic technique for chronic catheterization, and distalpunctures in a peripheral vein when multiple injections are required. Insome embodiments, the compound or composition can be delivered by rapidinjection into the vein using a syringe, administered intermittentlyover a specific amount of time using an IV secondary line, orcontinuously mixed in a main IV solution.

In other embodiments, a lentiviral vector is delivered using one ofvarious different types of catheters, including peripheral IV (PIV), anintravenous catheter inserted by percutaneous venipuncture into aperipheral vein for short-term IV therapy, or a central venous catheter(CVC) that is inserted into a large vein in the central circulationsystem. In some embodiments, the catheterization method used to deliverthe lentiviral vector includes IV infusion, pump infusion, dripinfusion, a tunneled catheter, an implanted port, and a peripherallyinserted central catheter.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions, or dispersions and/or sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In some embodiments, suitable carriers for intravenousadministration of a lentiviral vector include physiological saline,bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, N.J.), orphosphate buffered saline (PBS).

In all cases, the composition should be sterile and should be fluid tothe extent that it is easy to inject. In various embodiments, thepharmaceutical formulation is stable under the conditions of manufactureand storage and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. In general, the relevantcarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (including glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The proper fluiditycan be maintained by using a coating, such as lecithin, keeping therequired particle size (in the case of dispersions), and usingsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, including parabens,chlorobutanol, phenol, ascorbic acid and thimerosal. In someembodiments, isotonic agents, including sugars and polyalcohols, such asmannitol, sorbitol and sodium chloride, can be added to the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, suchas aluminum monostearate and gelatin (US 2005/0251872).

Sterile injectable solutions can be prepared by incorporating the activecompound (such as a lentiviral vector, or another expression vector) inthe required amount in an appropriate solvent with one, or acombination, of the ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof (US 2005/0251872).

Unless otherwise indicated, the practice of the present disclosure canemploy conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA. These methods aredescribed in the following publications. See, e.g., Sambrook, et al.Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M.Ausubel, et al. eds., Current Protocols in Molecular Biology (1987); theseries Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, etal., PCR: A Practical Approach, IRL Press at Oxford University Press(1991); MacPherson et al., eds. PCR 2: Practical Approach (1995); Harlowand Lane, eds. Antibodies, A Laboratory Manual (1988); and R. I.Freshney, ed. Animal Cell Culture (1987).

Sequence information provided by public database can be used to identifygene sequences to target and nucleic acid sequences encodingphenotype-altering proteins as disclosed herein. Exemplary sequences areprovided herein.

Variants of the sequences disclosed and referenced herein are alsoincluded. Variants of proteins can include those having one or moreconservative amino acid substitutions. As used herein, a “conservativesubstitution” involves a substitution found in one of the followingconservative substitutions groups: Group 1: Alanine (Ala), Glycine(Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp),Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine(Ile), Leucine (Leu), Methionine (Met), Valine (Val); and Group 6:Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function or chemical structure or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, andGln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser,Thr, Pro, and Gly; polar, negatively charged residues and their amides:Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg,and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, andCys; and large aromatic residues: Phe, Tyr, and Trp. Additionalinformation is found in Creighton (1984) Proteins, W.H. Freeman andCompany.

Variants of gene sequences can include codon optimized variants,sequence polymorphisms, splice variants, and/or mutations that do notaffect the function of an encoded product to a statistically-significantdegree.

Variants of the protein, nucleic acid, and gene sequences disclosedherein also include sequences with at least 70% sequence identity, 80%sequence identity, 85% sequence, 90% sequence identity, 95% sequenceidentity, 96% sequence identity, 97% sequence identity, 98% sequenceidentity, or 99% sequence identity to the protein, nucleic acid, or genesequences disclosed herein.

“% sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenprotein, nucleic acid, or gene sequences as determined by the matchbetween strings of such sequences. “Identity” (often referred to as“similarity”) can be readily calculated by known methods, including (butnot limited to) those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (Von Heijne, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) OxfordUniversity Press, NY (1992). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR, Inc., Madison, Wis.). Multiplealignment of the sequences can also be performed using the Clustalmethod of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) withdefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevantprograms also include the GCG suite of programs (Wisconsin PackageVersion 9.0, Genetics Computer Group (GCG), Madison, Wis.); BLASTP,BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990);DNASTAR (DNASTAR, Inc., Madison, Wis.); and the FASTA programincorporating the Smith-Waterman algorithm (Pearson, Comput. MethodsGenome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20.Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within thecontext of this disclosure it will be understood that where sequenceanalysis software is used for analysis, the results of the analysis arebased on the “default values” of the program referenced. As used herein“default values” will mean any set of values or parameters, whichoriginally load with the software when first initialized.

EXEMPLARY EMBODIMENTS

A first embodiment provides a method of treating a subject in need ofplasma Factor VIII, the method involving administering to the subject atherapeutically effective amount of an inflammation suppressor;administering to the subject a therapeutically effective amount of aCD8+ T cell depleting agent; and administering to the subject viaintraosseous (IO) infusion a therapeutically effective amount of acomposition including a lentiviral vector (LV) including an optimizedFVIII expression cassette expressibly linked to a megakaryocyte-specificpromoter. It is specifically contemplated that administration of theinflammation suppressor and/or the CD8+ T cell depleting agent may occurbefore, after, or concurrently with administration of the IO infusion,and may occur at the same or different times.

Notably, examples of such treatment methods do not involvepre-conditioning or myeloablative treatment of the subject.

The inflammation suppressor in examples of such methods is dexamethasone(Dex). By way of nonlimiting example, the inflammation suppressor (suchas dexamethasone) is administered in doses of 100 mg/kg at −24 h before,−4 h before, 4 h after, and 24 h after IO infusion of the LV.

The CD8+ T cell depleting agent in examples of the provided methodsincludes an anti-CD8 antibody, such as an anti-CD8α mAb. For instance,in some cases the anti-CD8α mAb is administered in doses of 4 mg/kg at−1 day before, 4 days after, and 11 days after IO infusion of the LV.

In any of the provided methods, the megakaryocyte-specific promoter maybe a GP1b-alpha promoter.

Generally, the IO infusion in provided treatment methods is carried outunder a controlled, relatively slow rate in order to facilitateeffective transduction by the provided LV of cells in the bone marrow.In specific example methods, the IO infusion is carried out at a rate of2 μL/min to 15 μL/min, or 5 μL/min to 12 μL/min, or at no more than 10μL/min. In additional examples, for instance when a relatively large(more than 2 kilograms) subject is being treated, the IO infusion iscarried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.Optimally in some embodiments, IO infusion is carried out over a periodof no more than 45 minutes.

Beneficially, the transgenic FVIII expressed from a LV deliveredintraosseously using a method provided herein is expressed in plateletsat a therapeutically relevant (that is, relatively high) level, and isfunctional in the subject being treated upon clotting cascade triggeringof the expressing platelets. By way of example, the optimized FVIIIexpression cassette includes the hFVIII variant hF8X10 K12 (SEQ IDNO: 1) or the hFVIII variant hF8/N6K12RH (SEQ ID NO: 2). Though in noway intended to be limiting, the lentiviral vector used in such methodscan include SEQ ID NO: 3 or SEQ ID NO: 4 (respectively), or a functionalvariant thereof which is capable of expressing the variant factor VIIIin platelets of the subject.

In any of the provided embodiments, the method of treating a subject inneed of plasma Factor VIII may be a method for treating a hemostasisrelated disorder in the subject, specifically wherein the subject is inneed of such treatment. By way of example, the subject receivingtreatment with methods described herein include subjects that havehemophilia A (hemA), hemophilia B (hemB), von Willebrand disease,bleeding associated with trauma or injury, thrombosis, thrombocytopenia,stroke, coagulopathy, disseminated vascular coagulation (DIC), orover-anticoagulation treatment disorder.

Specifically encompassed herein are treatment methods where the subjectis a HemA subject with preexisting anti-FVIII inhibitory antibodies. Theplatelet-limited hFVIII expression offered by Applicant's methods areparticularly beneficial to such subjects, as the expressed FVIII variantis not exposed and circulating in the blood and therefore does notelicit an immune response from circulating anti-FVIII inhibitoryantibodies.

The Exemplary Embodiments and Examples below are included to illustrateparticular embodiments of the disclosure. Those of ordinary skill in theart will recognize in light of the present disclosure that many changescan be made to the specific embodiments disclosed herein and stillobtain like or similar results without departing from the spirit andscope of the disclosure.

EXEMPLARY EMBODIMENTS

1. A method of treating a subject in need of plasma Factor VIII,including: administering to the subject a therapeutically effectiveamount of an inflammation suppressor; administering to the subject atherapeutically effective amount of a CD8+ T cell depleting agent; andadministering to the subject a therapeutically effective amount of acomposition including a lentiviral vector including an optimized FVIIIexpression cassette expressibly linked to a megakaryocyte-specificpromoter, wherein administration is via intraosseous (IO) infusion;intravenous (IV) delivery; or intranasal (IN) or inhaled delivery.2. The method of embodiment 1, wherein the inflammation suppressorincludes dexamethasone (Dex).3. The method of embodiment 2, wherein the dexamethasone is administeredin doses of 100 mg/kg at −24 h before, −4 h before, 4 h after, and 24 hafter administration of the LV.4. The method of embodiment 1, wherein the CD8+ T cell depleting agentincludes an anti-CD8 antibody.5. The method of embodiment 4, wherein the CD8+ T cell depleting agentis an anti-CD8α mAb administered in doses of 4 mg/kg at −1 day before, 4days after, and 11 days after delivery of the LV.6. The method of embodiment 1, wherein the megakaryocyte-specificpromoter is a GP1b-alpha promoter.7. The method of embodiment 1, which method does not includepre-conditioning or myeloablative treatment of the subject.8. The method of embodiment 1, wherein the IO infusion is carried out ata rate of 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at no morethan 10 μL/min.9. The method of embodiment 1, wherein the IO infusion is carried out ata rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.10. The method of embodiment 1, wherein the IO infusion is carried outover a period of no more than 45 minutes.11. The method of embodiment 1, wherein the optimized FVIII expressioncassette includes hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH (SEQ ID NO:2).12. The method of embodiment 1, wherein the lentiviral vector includesSEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant thereof.13. The method of embodiment any one of the previous embodiments, whichis a method for treating a hemostasis related disorder in the subject,and the subject is in need of such treatment.14. The method of any one of the previous embodiments, wherein thesubject has hemophilia A (hemA), von Willebrand disease, bleedingassociated with trauma or injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated vascular coagulation (DIC), orover-anticoagulation treatment disorder.15. The method of any one of the previous embodiments, wherein thesubject is a HemA subject with preexisting anti-FVIII inhibitoryantibodies.16. A method of treating a subject in need of plasma Factor VIII,essentially as described herein.

Example 1. Enhancing Therapeutic Efficacy of In Vivo Platelet-TargetedGene Therapy of Hemophilia A

Previously described in vivo hematopoietic stem cell (HSC)-based,platelet-targeted gene therapy protocols partially corrected thebleeding phenotype in hemophilia A (HemA) mice for over five months. Inthat protocol, lentiviral vectors (LVs) carrying a factor III (FVIII)transgene (G-F8/N6-LV), driven by a megakaryocyte-specific promoter(Gp1bα), were delivered via intraosseous (IO) infusion to transduceHSCs. Maturation of transduced HSCs down the megakaryocyte-lineage ledto ectopic FVIII production in platelets. However, an effective andclinically feasible protocol was still required to improve LVtransduction efficiency and increase platelet-FVIII functionality.

To enhance LV transduction, this example describes a combined drugregimen of dexamethasone and anti-CD8α monoclonal antibody, followed byIO infusion of LVs in mice. In M-GFP-LV-treated (M=MND: ubiquitouspromoter) C57BL/6 mice, a 2-fold increase in GFP+ bone marrow cellnumbers was observed on day 7 and up to 14% of GFP+ HSCs on day 160. InG-F8/N6-LV treated HemA mice, significant improvement in phenotypiccorrection was seen on day 84.

In addition, to improve platelet-FVIII functionality, F8X10K12 (withincreased expression and bioactivity compared to F8/N6) was incorporatedinto the LVs. Treatment with G-F8X10K12-LV in HemA mice produced ahigher level of platelet-FVIII, however induced high-titer anti-FVIIIinhibitors. This example also shows incorporation of F8/N6K12RH withenhanced bioactivity only to LVs. Following treatment with combineddrugs and IO infusion of G-F8K12RH-LV, HemA mice showed significantphenotypic correction without anti-FVIII inhibitor formation. Thisprotocol may provide a readily translatable treatment for hemophilia.

Thus, here, an effective and clinically feasible protocol was developedby improving LV transduction efficiency and increasing platelet-FVIIIfunctionality. To enhance LV transduction, a combined drug regimen ofdexamethasone and anti-CD8α monoclonal antibody was administered,followed by IO infusion of LVs. In M-GFP-LV-treated (M=MND: ubiquitouspromoter) C57BL/6 mice. A 2-fold increase in GFP+ bone marrow cellnumbers was observed on day 7 and up to 14% of GFP+ HSCs on day 160. InG-F8/N6-LV treated HemA mice, significant improvement in phenotypiccorrection was seen on day 84. To improve platelet-FVIII functionality,F8X10K12 with increased expression and bioactivity compared to F8/N6,was incorporated into the LVs. Treatment with G-F8X10K12-LV in HemA miceproduced a higher level of platelet-FVIII, however induced high-titeranti-FVIII inhibitors. Further, following treatment with combined drugsand IO infusion of G-F8K12RH-LV (which has enhanced bioactivity), HemAmice showed significant phenotypic correction without anti-FVIIIinhibitor formation. This protocol may provide a readily translatabletreatment for hemophilia.

Introduction.

Inhibitory antibody formation against human factor VIII (FVIII) is asignificant complication in treatment of hemophilia A (HemA) patients.Ectopic expression of FVIII in platelets could be an effective approachfor treating HemA. Unlike circulating plasma FVIII, FVIII stored inα-granules of platelets is protected from being processed by antigenpresenting cells, therefore significantly reducing the chance to induceanti-FVIII immune responses. Moreover, platelet FVIII is not neutralizedby preexisting anti-FVIII inhibitory antibodies (inhibitors), which willbenefit HemA patients who already developed inhibitors. During bleeding,the activated platelets locally excrete FVIII that can directlyparticipate in the coagulation cascade and promote clot formation. Ithas recently been demonstrated that low levels of platelet FVIII canpartially correct hemophilia phenotype in animal models.

One approach to direct FVIII long-term expression in platelets isintraosseous (IO) infusion of lentiviral vectors (LVs) carrying a FVIIItransgene controlled by a megakaryocyte specific promoter Gp1bα(Gp1bα-F8-LVs) (Wang et al., Mol Therapy 23:617-626, 2015). In this invivo gene therapy protocol of HemA, hematopoietic stem cells (HSCs) wasefficiently transduced by LVs in situ, followed by FVIII transgeneexpression in the late stage of megakaryocyte differentiation, andfinally storage of FVIII protein in α-granules of platelets. Previouswork showed that a single IO infusion of Gp1bα-F8-LV resulted in over 3%platelets containing hFVIII, leading to partial phenotype correction inimmune competent HemA mice with and without pre-existing inhibitors.

Furthermore, in vivo delivery of LVs can avoid many difficulties andpotential toxicities encountered by ex vivo gene therapy including lossof stem cell properties and engraftment potential after cell transfer,negative effects of ex vivo cytokine stimulation and pre-conditioning ofthe subject. However, one potential limitation of in vivo gene therapyprotocols is the induction of LV-specific immune responses, which maydecrease LV transduction efficiency and eliminate LV transduced cells.It has been shown that dexamethasone (Dex) can suppress inflammatoryresponses after intravenous delivery of L Vs into the immune competentmice and increase LV transduction efficiency. It has also been shownthat Rapamycin (Rapa) can enhance LV transduction efficiency for both invivo and ex vivo gene therapy.

An optimized transgene for effective in vivo gene therapy of hemophiliawould confer a higher gene expression level and a transgene product withenhanced biological activity. Since the size of a full-length humanFVIII cDNA is quite large (>7 kbp), it cannot be easily packaged inviral vectors, often resulting in low titers of viruses. Thereforeshorter cDNAs coding for FVIII variants were used in gene therapypreclinical research. Importantly, a B-domain deleted FVIII (BDD-FVIII)variant exhibits similar functional activity as the full-length FVIII(Pittman et al., Blood 81:2925-2935, 1993). Additional mutations werealso incorporated into the gene coding for the BDD-FVIII to increase itssecretion, such as, F8/N6 with a 226 aa B-domain variant sequence (Miaoet al., Blood 103:3412-3419, 2004), codon optimized BDD-FVIII and F8/N6(Ward et al., Blood 117:798-807, 2011), F8-V3 (McIntosh et al., Blood121:3335-3344, 2013) with a 17-aa peptide coding sequence replacing the226 aa/N6 in F8/N6, and F8-RH encoding a BDD-FVIII variant with R1645H(Siner et al., Blood 121:4396-4403, 2013). Some FVIII variants, likeFVIII-RH and furin-cleavage site deleted BDD-FVIII variants (Nguyen etal., J Thromb Haemostas. 15:110-121, 2017), exhibit an increase inbiological activity compared with BDD-FVIII, likely due to its slowerdissociation of the A2-domain upon thrombin activation.

In this example, the therapeutic efficacy of HemA gene therapy wasimprove using IO infusion of LVs targeting FVIII expression inplatelets. Immune competent mice were pretreated with Dex to suppressinflammatory responses and anti-CD8α monoclonal antibody (mAb) toinhibit cytotoxicity by transient depletion of CD8⁺ T cells. Thepharmacologic intervention with combined Dex and anti-CD8 mAb treatmentimproved LV transduction efficiency and increased long-term transgeneexpression levels in mice. Furthermore, two new human FVIII (hFVIII)variants with higher expression and enhanced biological activity werealso tested in immune competent HemA mice. The results demonstrated thatcombined drug treatment plus IO infusion of LVs containing the FVIIIvariant gene with enhanced biological activity significantly improvedhemophilia phenotype correction.

Materials and Methods

Animals: All mice were kept in a specific pathogen-free environment atSeattle Children's Research Institute (SCRI) according to NationalInstitutes of Health guidelines for animal care and the guidelines ofSCRI. The protocols were approved by the Institutional Animal Care andUse Committee at SCRI. HemA mice (factor VIII exon 16 knockout) withC57BL/6 (BL/6) genetic background were generated by crossing the mixedbackground HemA mice (SV129/BL6) with BL/6 mice for eight generations(Miao et al., Blood 114:4034-4044, 2009). BL/6 mice were purchased fromthe Jackson Laboratory. Only male HemA mice were used in this study.

Human factor VIII cDNA variants: Three human factor VIII cDNA variantswere incorporated into the LVs, including B-domain variant of humanfactor VIII (hF8/N6) (Pipe, Haemophilia 15:1187-1195, 2009), hF8X10K12(complete lentiviral construct: SEQ ID NO: 3), and hF8/N6K12RH (completelentiviral construct: SEQ ID NO: 4). The sequences of the hF8X10K12 andhF8/N6K12RH inserts are provided in SEQ ID NOs: 1 and 2, respectively.Human F8 cDNA variants: Three human F8 cDNA variants were incorporatedinto the LVs, including B-domain variant of hFVIII (hF8/N6)22,hF8X10K1223,24 and hF8/N6K12RH19,24 (FIG. 1a ).

Intraosseous infusion of lentiviral vectors: Lentiviral vectors weredelivered into the mice by IO infusion as in previous studies (Wang etal., Mol Therapy 23:617-626, 2015; Wang et al., Blood 124:913-923, 2014;both incorporated herein by reference). Detailed information about theconstruction and production of lentiviral vectors and IO infusion oflentiviral vectors is provided below.

Immunosuppression treatment: Dex (MWIVet) and anti-CD8α mAb (BioXcell;clone YTS169.4) were used to transiently deplete innate immune responsesand cytotoxicity, respectively. LVs were infused into the mice on day 0.Single drug pretreated mice were given Dex (100 mg/kg, −24 h, −4 h, 4 hand 24 h) or anti-CD8α mAb (4 mg/kg, day −1, 4 and 11) byintraperitoneal injection. Combined drugs pretreated mice wereadministrated with Dex (100 mg/kg, −24 h, −4 h, 4 h and 24 h) andanti-CD8α mAb (4 mg/kg, day −1, 4, 11, 16 and 21).

Characterization of transgene GFP/hFVIII expression: For M-GFP-LVtreated mice, GFP expression in bone marrow cells of M-GFP-LV3 (LVcarrying a GFP gene driven by the modified myeloid proliferative sarcomavirus (MND) promoter (Astrakhan et al., Blood 119:4395-4407, 2012;Challita et al., J. Virol. 69:748-755, 1995); FIG. 7B) treated BL6 miceand hFVIII expression in F8-LV transduced 293T cells were detected byflow cytometry. Flow cytometric analysis was conducted using a FACSLSRII (BD Biosciences) and the data were analyzed using FlowJo software(version 8.8.1; Tree Star). hFVIII expression in platelets ofF8-LV-treated HemA mice was measured by ELISA, as previously described(Wang et al., Mol Therapy 23:617-626, 2015). The detailed process toisolate bone marrow cells, white blood cells and platelets and thedetailed information about the antibodies used in this study areprovided below.

Assays for measuring hFVIII activities and anti-hFVIII antibodies:Plasma samples were isolated from the peripheral blood in experimentalmice, which was obtained by retro-orbital bleeding. hFVIII activitieswere analyzed using a modified activated partial thromboplastin timeassay (aPTT), and anti-hFVIII antibodies were measured by hFVIIIBethesda inhibitor assay as previously described (Miao et al., Blood114:4034-4044, 2009; Kasper & Aronson, Thromb Diath Haemorrp 34:612,1975).

Assays to characterize phenotypic correction in HemA mice: Correction ofthe bleeding phenotype of G-F8-LV treated HemA mice was evaluated usinga modified tail clip assay, as previously described (Wang et al., MolTherapy 23:617-626, 2015). The function of platelet-stored hFVIII in LVtreated HemA mice was evaluated by Thrombin generation assay (TGA) usinga TECHNOTHROMBIN® TGA Kit (Technoclone GmbH, Austria) and rotationalthromboelastometry (ROTEM®) assay. The blood flow rate of the rightcarotid artery in G-F8-LV treated HemA mice after local damage wasmeasured in a ferric chloride (FeCl₃)-induced thrombosis model. Thedetailed procedures of TGA, ROTEM, the FeCl₃ injury model are describedbelow.

Statistical analysis: Data were expressed as mean ±the standard error ofthe mean (SEM). The statistical significance of the difference betweenmeans was determined using two-sample assuming unequal variances t test.Differences were considered significant at P less than 0.05.

Antibodies: Mouse hematopoietic lineage flow cocktail (Lineage) eFluor®450, allophycocyanin (APC) anti-mouse CD117 (C-Kit) andPhycoerythrin-Cy7 (PE-Cy7) anti-mouse Ly-6A/E (Sca-1) were purchasedfrom eBioscience. A polyclonal sheep anti-human factor VIII (SAF8C) wasfrom Affinity Biologicals INC. A rabbit anti-sheep IgG antibodyhorseradish peroxidase (HRP) was from Bio-Rad Laboratories. Mouseanti-human factor VIII monoclonal antibody ESH-8 and GMA-012 wereobtained from American Diagnostica Inc. and Green Mountain Antibodies,respectively. Goat anti-mouse Ig FITC was from BD BiosciencesPharMingen.

Lentiviral vector construction, production and titration: BothpRRL⋅SIN⋅Gp1bα⋅hF8/N6⋅WPRE (pGp1bα-F8/N6) and pRRL⋅SIN·EF1α⋅hF8/N6·WPRE(pEF1α-F8/N6) were cloned in previous work (Wang et al., Mol Therapy23:617-626, 2015). Then hF8/N6 in pGp1bα-F8/N6 and pEF1α-F8/N6 wasreplaced by hF8X10K12 and hF8/N6K12RH to createpRRL⋅SIN⋅Gp1bα⋅hF8X10K12⋅WPRE (pGp1bα-F8X10K12) andpRRL⋅SIN⋅Gp1bα⋅hF8X/N6K12RH⋅WPRE (pGp1bα-F8/N6K12RH), andpRRL⋅SIN⋅EF1α⋅hF8X10K12⋅WPRE (pEF1α-F8X10K12) andpRRL⋅SIN⋅EF1α⋅hF8X/N6K12RH⋅WPRE (pEF1α-F8/N6K12RH), respectively.pRRL⋅SIN⋅MND⋅eGFP⋅WPRE was obtained from the virus core at SCRI. All thereagents for cloning and isolating plasmids were purchased from Qiagen.The LVs (G-F8/N6-LV, G-F8X10K12-LV, G-F8/N6K12RH-LV, E-F8/N6-LV,E-F8X10K12-LV, and M-GFP-LV) were produced by the transient transfectionof human embryonic kidney (HEK) 293T cells using polyethylenimine (PEI)and three plasmids including one transgene-LV construct, pMD2.G andpAX2G. Viral titers (ifu/ml) were determined by real-time quantitativePCR (qPCR) as described in previous studies (Wang et al., Mol Therapy23:617-626, 2015; Auti et al., Embo Mol Med. 9:737-740, 2017).

HEK 293T cell transduction: HEK 293T cells were maintained in DMEM(Corning Cellgro) with 10% fetal bovine serum (FBS, AtlanticBiologicals), 2 mM L-glutamine (Corning Cellgro), 10 mM HEPES (CorningCellgro) and 100 IU/ml Penicillin/100 μg/ml Streptomycin (CorningCellgro). 5×10⁴ cells were cultured in 200 μL medium and transduced withE-F8/N6-LV (MOI=100) and E-F8X10K12-LV (MOI=100) overnight with 4 μg/mlpolybrene. After 24 h, 200 μL of culture medium was added. Cell werecultured for another 96 h, and collected to check FVIII expression via aflow cytometer (LSRII, BD Biosciences).

Intraosseous infusion of lentiviral vectors: The detailed intraosseous(IO) infusion procedure was described in the previous study (Wang etal., Mol Therapy 23:617-626, 2015). Briefly, the mouse was anesthetizedwith 2.5% isoflurane, and the hair around the right knee was shaved andthe skin was sterilized and excised to expose the muscle layer. Aftertwisting and carefully inserting a sterile 27-gauge needle fixed with aninsulin syringe into the right tibia through the joint, 20 μL LVs wasinfused into the tibia at a speed of 10 μL/min via a new sterile27-gauge needle connected with a microliter syringe (Hamilton). Theinfusion speed and volume were precisely controlled by a pump (NE-1010,New Era Pump Systems). The cut site was sutured after infusion.

Detailed methods of isolating bone marrow cells, white blood cells andplatelets: Bone marrow cells were collected by flushing both femur andtibia of the mice with 5 ml of RPMI 1640. After lysing red blood cellswith Ammonium-Chloride-Potassium (ACK) lysing buffer (0.15 M NH₄Cl, 10mM KHCO₃, 1 mM EDTA, pH 8.1), other cells were obtained after filteringthe medium through a filter with the pore size of 0.45 μm (FisherScientific). White blood cells and platelets were isolated fromheparinized blood, which was collected by retro-orbital bleeding. Whiteblood cells were collected after lysing red blood cells with ACK buffer.For platelets, 140 μL of heparinized blood was added with 200 μL of RPMI1640, and the mixture was carefully transferred to the top of 300 μL ofFico/Lite™-Platelets (Atlanta Biologicals) and spin at 100 g for 10 min.Then carefully removed the clear upper solution and collected the cloudymiddle layer to spin at 1,000 rpm for 5 min. Small white plateletpellets were washed by FACS buffer (PBS, 5% bovine serum albumin) twice.

Real-time qPCR: Real-time qPCR was performed on genomic DNA (gDNA),which was extracted from the isolated white blood cells and bone marrowcells in experimental mice using DNeasy Blood & Tissue Kit from Qiagen.gDNA was subject to real-time qPCR using two primer/probe sets (one forintegrated lentiviral vector sequence and one for murine housekeepingβ-actin sequence) and TaqMan Universal PCR Master Kit (AppliedBiosystems) and run on Step One Plus real-time PCR System (AppliedBiosystems). Murine copy number was determined by threshold cycle, whichwas compared to standard curves generated using gDNA isolated fromsingle lentiviral copy A20 cell clones.

Detailed methods of thrombin generation assay: Firstly, both theprotocol for Calibration Curve measurement and the protocol for samplemeasurement were created in PerkinElmer® Wallac Manger. Then the wholeblood was drawn from the inferior vena cava (IVC) of the anesthetizedmice into 3.8% sodium-citrate solution. Platelet-rich plasma (PRP) wasobtained by centrifuging for 5 minutes at 100×g and carefully pipettingthe top layer. Platelet-poor plasma (PPP) from untreated HemA mice wascollected by centrifuging PRP for 10 minutes at 1,500 g. Plateletconcentration in PRP was measured by a qNaono (iZON Science). 30 μL ofPRP samples mixed with 10 μL of HemA PPP was added into black NUNCMaxisorp (Fisher). The plate was read immediately in a Victor 3instrument (Perkin-Elmer) after adding 50 μL of TGA substrate (SUB). Ina separate calibration measurement, 40 μL of thrombin calibrator at fourdifferent dilutions (1:2, 1:4, 1:20, 1:200) were combined with 50 μL TGASUB and thrombin generation was detected for 10 min in 30 sec intervalsat 37° C. Lag phase time, peak thrombin and the area under the curve(AUC) were obtained using the evaluation software provided byTechnoclone.

Blood flow rate measurement: The experimental mice were anesthetized by2.5% isoflurane. An incision was made with a scalpel directly over theright common carotid artery, and a segment of the artery was exposedusing blunt dissection. A miniature Doppler flow probe (Model 0.5 VB,Transonic System Inc., Ithaca, N.Y., USA) was attached to the carotidartery to monitor blood flow. Blood vessel damage was induced byapplying two pieces of filter paper (1×2 mm) saturated with 20% Ferricchloride (FeCl₃) solutions. The pieces of filter paper were placed onthe top of carotid artery in contact with the adventitial surface of thevessel. After 2-min exposure, the filter paper was removed and thevessel was washed with sterile normal saline. Carotid blood flow wascontinuously monitored for 30 min after FeCl₃ application. Wild-typeC57BL/6J mice and HemA mice were used as positive and negative controls,respectively. Average blood flow rate over 30 minutes after FeCl₃treatment was used to evaluate the extent of protection from occlusion.The mice were sacrificed after measurement.

Results

Comparison of GFP expression in immunocompetent and deficient micefollowing IO delivery of M-GFP-LV To evaluate if immune responses impacttransgene expression, M-GFP-LV (1.1×10⁸ infectious units (ifu)/animal)was intraosseously delivered into immune deficient Rag2^(−/−) mice andimmune competent BL6 mice. 7 days after infusion, GFP expression levelsin total bone marrow cells and HSCs in Rag2^(−/−) mice were similar tothat in BL6 mice (FIG. 1). Subsequently, GFP levels were increased inRag2^(−/−) mice but decreased in BL6 mice from day 7 to 28. In the longterm, 84 days after infusion, the GFP levels in both total bone marrowcells and HSCs of Rag2^(−/−) mice were significantly higher than thosein BL6 mice (FIG. 1). Overall, following M-GFP-LV transduction in bonemarrow, GFP expression maintained relatively stable at high levels inRag2^(−/−) mice, whereas GFP expression levels gradually decreased overtime in immune competent BL6 mice. These results suggest that immuneresponses elicited by LVs, likely including cytotoxic lymphocytes(CTLs), may reduce the number of transduced cells over time.

Pharmacological approach to enhance GFP expression in immune competentmice following with IO delivery of M-GFP-LV Based on the resultsobtained from LV-treated immune-competent and deficient mice, whetherpharmacological agents designed to non-specifically or specificallyblock these immune responses will limit clearance of transduced cells,leading to more persistent and higher levels of sustained transgeneexpression, was explored. Firstly, BL6 mice were treated Dex with (100mg/kg, −24 h, −4 h, 4 h and 24 h) before and after IO infusion ofM-GFP-LV (8.8×10⁸ ifu/animal, day 0). 7 days after LV infusion, theDex+LVs treated mice produced significantly higher GFP expression levelsin both total bone marrow cells and HSCs than those in LVs only treatedmice (FIG. 2B), indicating that Dex could efficiently inhibit earlyimmune responses and enhance GFP expression. Moreover, successful LVtransduction was indicated by LV copy numbers in total bone marrow cellsof Dex+LVs and LVs only treated mice to be 0.90 and 0.80, respectively.

Secondly, CD8⁺CD3⁺ T cells were transiently depleted by IP injection ofanti-CD8α mAb to suppress cytotoxic function (FIG. 8A). The mice weretreated with anti-CD8α mAb (4 mg/kg, day −1, 4 and 11) before and afterIO infusion of M-GFP-LV (1.1×10⁸ ifu/animal). 9 days after IO infusion,GFP expression levels in total bone marrow cells and HSCs in anti-CD8αmAb+LVs treated mice were significantly higher than those in LVs onlytreated mice (FIG. 2C). 30 and 63 days later, although GFP expressionlevels decreased over time, the levels in anti-CD8ζ mAb+LVs treated micewere higher than that in LVs only treated mice (FIG. 2C and FIG. 9).CD8α⁺CD3ε⁺ T cells were totally depleted in blood and bone marrow intreated mice 4 days after anti-CD8α mAb treatment (day 3 after IOinfusion), then recovered gradually to normal levels (FIG. 8B). Thus,GFP expression in anti-CD8α mAb+LVs treated mice was partially rescuedby transient depletion of CD8α⁺CD3ε⁺ T.

In order to increase LV transduction and preserve survival of transducedcells, a short course of combination drug treatment with Dex (100 mg/kg,−24 h, −4 h, 4 h and 24 h), and anti-CD8α mAb (4 mg/kg, day −1, 4, 11,16 and 21) before and after IO infusion of M-GFP-LV (3.6×10⁸ ifu/animal)was administered. 7 days after IO infusion, GFP expression levels intotal bone marrow cells in drugs+LVs treated mice was significantlyhigher than that in LVs only mice. Whereas, GFP expression level in HSCswas similar in these two groups of mice probably because HSCtransduction reached saturation with relatively high dose of LVs. In thelong term, GFP expression levels in both total bone marrow cells andHSCs in Drugs+LVs treated mice were significantly higher than that inLVs only treated mice (FIG. 2D and FIG. 10). Furthermore, lentiviralcopy numbers in blood cells and total bone marrow cells were detectablein both groups of mice with slightly higher number in drugs+LVs treatedmice than that in LVs treated mice on day 160 (FIG. 2E). These resultsindicated that transient administration of combined drugs partiallysuppressed immune responses and over 10% of GFP⁺HSCs were achieved overfive months after IO infusion of M-GFP-LV.

Pharmacological approach to improve phenotype correction in immunecompetent HemA mice with IO delivery of G-F8/N6-LV. Next, the samepharmacological approach was applied to improve the therapeutic effectsof IO infusion of G-F8/N6-LV in HemA mice. HemA mice were given with thecombined drug treatment (Dex+anti-CD8α mAb) plus IO infusion ofG-F8/N6-LV (2.2×10⁶ ifu/animal). 10 weeks after IO infusion, hemophiliaphenotype correction was evaluated with two injury mouse models: tailclipping and FeCl₃-induced thrombosis. In the tail clipping assay, theLVs only or drugs+LVs treated mice had significantly reduced blood lossthan HemA control mice (FIG. 3A). In the FeCl₃-induced carotid arteryinjury model, the average blood flow rates in the LVs only or Drugs+LVstreated mice were significantly slower than that in HemA control mice(FIG. 3B), confirming that IO infusion of G-F8/N6-LV into HemA mice canenhance their blood clotting function. Furthermore, the drugs+LVstreated mice lost noticeably less blood than the LVs only treated micein tail clipping assay (FIG. 3A). Consistent with that, the averageblood flow rate in Drugs+LVs treated mice was slower than that in LVsonly treated mice (FIG. 3B). These results indicated that combined drugtreatment with Dex and anti-CD8α Ab could significantly enhance thetherapeutic efficiency of IO infusion of G-F8/N6-LV in HemA mice. Inaddition, there was neither detectable plasma FVIII activity noranti-FVIII inhibitors in blood (FIG. 3C), indicating that no detectableFVIII secretion into blood.

F8X10K12 with higher expression and higher functional activity combinedwith drug treatment improved hemophilia A phenotypic correction. Toimprove therapeutic effects of in vivo gene therapy of HemA mice, cDNAsencoding FVIII variants with high expression and functional activitywere next incorporated. F8X10K12 was a novel human FVIII cDNA variantwith a deleted B-domain, mutations in A1 domain to enhance secretion(X10), and mutations in Cl and C2 domains to increase functionalactivity (K12) (FIG. 4A). Its expression was tested by hydrodynamicinjection of the plasmid, pEF1α-F8X10K12, into HemA mice. Compared withpEF1α-F8/N6, pEF1α-F8X10K12 produced a dramatically high levels of FVIIIin treated mouse plasma on day 4 (FIG. 4B). E-F8X10K12-LV was also madeto transduce 293T cells (M01=100). Higher percentage and MFI of FVIIIgene expression were detected in E-F8X10K12-LV transduced 293T cellsthan in E-F8/N6-LV transduced cells on day 4 (FIG. 4C), confirming thatF8X10K12 enhanced FVIII production following LV transduction.

Next, G-F8X10K12-LVs (FIG. 5A) was generated and delivered into HemAmice with combined drug treatment. The average platelet-hFVIII antigenlevels in G-F8X10K12-LV+drugs and G-F8X10K12-LV only treated mice weresignificantly higher than that in G-F8/N6-LV treated mice on day 90(FIG. 5B), suggesting that F8X10K12 produced higher platelet-FVIIIlevels. No anti-FVII I antibody was detected in G-F8/N6-LV treated miceand 8 of 11 G-F8X10K12-LV+drugs treated mice (FIG. 5C), whereas allG-F8X10K12-LV only treated mice (n=9) and 3 of 11G-F8X10K12-LV+drugs-treated mice generated anti-hFVIII antibodies (FIG.5C). Judging from the fact that the average platelet-hFVIII antigenlevels in G-F8X10K12-LV+drugs or G-F8X10K12-LV only treated mice weresimilar, it was suspected that significant portions of the FVIIIproduced in G-F8X10K12-LV treated mice were secreted into the plasma dueto the enhanced expression and secretion of F8X10K12 protein, thusfacilitating the generation of anti-FVIII antibodies. These results alsosuggested that combined drug treatment could suppress anti-FVIII humoralimmune responses in G-F8X10K12-LV+drugs treated mice. Viral copy numberwas detected in blood cells of all treated mice on day 120 (FIG. 5D),indicating that LVs successfully transduced primitive HSCs.

The long-term therapeutic efficiency of combined drugs+LVs in HemA micewas evaluated by a tail clipping assay. On day 120 after IO infusion ofLVs, all three groups of mice treated with F8X10K12-LV+drugs, orG-F8X10K12-LV only, or G-F8/N6-LV only, showed significantly reducedblood loss compared with HemA control mice (FIG. 5E), indicating that IOinfusion of all three vectors can correct bleeding phenotype in HemAmice. Furthermore, minor differences were detected in the blood losswithin these three groups with the trend of G-F8X10K12-LV+drugs<G-F8X10K12-LV only <G-F8/N6-LV only treated mice (FIG. 5E).

The functional activity of platelet-FVIII was also evaluated in vitro bythrombin generation assay using PRP isolated from HemA mice. Althoughplatelets play a major role in the burst of thrombin generation inclotting cascade, FVIII stored in platelets enhances this process topromote fibrin clot formation. Compared to HemA controls, wild-typecontrols had shorter lag phase time, higher peak height thrombin andhigher total generated thrombin (area under the curve (AUC) (FIG. 5F).The average lag phase time of all PRP samples from the treated mice onday 200 after IO infusion of LVs was shorter than that of HemA controls,which confirmed that platelet-FVIII played an important role in theearly stage of clot formation. In addition, the average peak heightthrombin and total generated thrombin of samples fromG-F8X10K12-LV+drugs treated mice were relatively higher than that fromG-F8/N6-LV only treated mice, indicating that the mice treated withimmune suppressive drugs and F8X10K12-LV generated more thrombin toenhance clot formation.

F8/N6K12RH with higher functional activity combined with drug treatmentsignificantly enhanced hemophilia A phenotyping correction Even thoughrelatively higher platelet-FVIII level in G-F8X10K12-LV treated HemAmice enhanced blood clotting, high-titer inhibitory antibodies wereinduced due to secretion of FVIII into the circulation. Thus F8/N6RHK12(FIG. 6A) was cloned into the LV to decrease secretion and maintainhigher functional activity and stability of FVIII protein. As shown inearlier sections, F8/N6 is a B-domain deleted FVIII variant that canenhance FVIII gene expression without induction of inhibitory anti-FVIIIantibodies (FIGS. 3 & 5). RH in F8/N6RHK12 denotes R1645H mutation(Siner et al., Blood 121:4396-4403, 2013) for generating a more stableFVIII single chain molecule and together with K12 mutations cansignificantly enhance the biological activity of FVIII compared withBDD-FVIII. As expected, the mice treated with hydrodynamic injection ofpEF1α-F8/N6RHK12 produced significantly higher plasma FVIII activitythan the mice treated with pEF1α-F8/N6 (FIG. 6A).

G-F8/N6K12RH-LV was generated and delivered into HemA mice with combineddrug treatment. As expected, the average platelet-FVIII antigen levelsin G-F8/N6K12RH-LV-only and G-F8/N6-LV-only treated mice were similar.However, the platelet-FVIII levels in G-F8/N6K12RH-LV+drugs-treated micewere significantly higher than that in G-F8/N6K12RH-LV-only andG-F8/N6-LV-only treated mice on day 84 (FIG. 6C), suggesting that thecombined drug treatment enhanced the production and maintenance ofhigher platelet-FVIII levels. There was neither detectable plasma FVIIIactivity nor anti-FVIII inhibitors in blood of the treated mice on day84 (FIG. 6C). Phenotype correction in the treated mice was nextevaluated by the blood flow rate change in the FeCl₃-induced carotidartery injury mouse model. The average flow rates were compared in fivemouse groups: untreated wild-type G-F8/N6K12RH-LV+drugs<G-F8/N6K12RH-LVonly <G-F8/N6-LV only treated<untreated HemA mice on day 84 (FIG. 6D)with several of G-F8/N6K12RH-LV+drugs-treated mice exhibiting the sameflow rates as observed in wild-type mice. The function of platelet FVIIIin these five groups of mice was also evaluated using ROTEM assay. Theresults were consistent with the flow rate data. Interestingly, themaximum clot firmness (MCF) was easily corrected by platelet FVIII,whereas it is more difficult to correct the clotting time (CT).

HemA phenotype correction of G-F8/N6-LV only or G-F8/N6K12RH-LV+drugstreated mice was also evaluated by measuring carotid artery blood flowrate on day 84 (FIG. 6D). Phenotype correction in the treated mice wasnext evaluated by the blood flow rate change in the FeCl₃-inducedcarotid artery injury mouse model. The average flow rates were comparedin five mouse groups: untreated wild-typeG-F8/N6K12RH-LV+drugs<G-F8/N6K12RH-LV only<G-F8/N6-LV onlytreated<untreated HemA mice on day 84 (FIG. 6D) with several ofG-F8/N6K12RH-LV+drugs-treated mice exhibiting the same flow rates asobserved in wild-type mice. The function of platelet FVIII in these fivegroups of mice was also evaluated using ROTEM assay. The results wereconsistent with the flow rate data (Table 1). Interestingly, the maximumclot firmness (MCF) was easily corrected by platelet FVIII, whereas itis more difficult to correct the clotting time (CT).

TABLE 1 α MCF CT (s) CFT (s) (degree) (mm) G-F8/N6K12RH- 146 ± 4*  104 ±21  70 ± 4** 66 ± 4  LV + Drugs G-F8/N6K12RH-LV 388 ± 193 165 ± 123  62± 15* 61 ± 7  G-F8/N6-LV 564 ± 367 261 ± 182  43 ± 13  62 ± 6  Wild-Type164 ± 49* 57 ± 10* 79 ± 2** 63 ± 1  HemA  956 ± 185* 2370 ± 1150*  4 ±6*** 26 ± 19*

The whole blood samples were collected on day 140 to evaluate theplatelet FVIII function using a ROTEM assay. Each assay was carried outfor 1 hr to obtain values of clotting time (CT), clot formation time(CFT), α-angle, and max clot firmness (MCF). Statistical significancewas analyzed based on the relevance to the G-F8/N6-LV treated mousegroup. *<0.05, **<0.01, ***<0.001.

Discussion. Current clinical trials for hemophilia A or B gene therapyusing recombinant adeno-associated viral (AAV) vectors (rAAV) have shownvery promising results. However, lentiviral gene therapy is superior inseveral aspects. First, AAV persists as episomal, concatemerized vectorfollowing in vivo gene transfer. Overtime, transgene expression maydecrease, and repeated dosing will be required. It remains to be shownwhether repeated dosing is feasible, or effective. In contrast,lentiviral vectors are an integrating vector that is incorporated intothe genome of transduced cells. If successfully integrated intolong-lived bone marrow HSCs, a single treatment of LV-mediated genetherapy may be sufficient for life-long therapeutic benefit. Inaddition, In contrast to AAV which has a transgene capacity of 4.5 kb,LV can easily accommodate much larger genes such as FVIII cDNA. LV canefficiently transduce both dividing and non-diving cells, leading toefficient transduction of primitive HSCs. Furthermore, recent clinicaltrials of ex vivo HSC gene therapy have shown remarkable therapeuticbenefits and safety records for patients with severe genetic diseasesincluding X-linked severe combined immunodeficiency, Wiskott-Aldrichsyndrome, spinal muscular atrophy, and sickle cell disease Mostsignificantly, genotoxicity by clonal dominance or significant clonalexpansions from vector integration were not observed in these patients.

In the current study, the developed gene therapy protocol for HemA aimsto treat HemA patients by IO infusion of LVs containing FVIII cDNA underexpression control by a megakaryocyte-specific promoter, which leads toexpression of the gene in the platelets. This gene therapy protocol hasseveral advantages over existing protocols that include the ex vivo genetherapy protocol targeting HSC transduction or the in vivo gene therapyprotocol targeting FVIII expression in hepatocytes. Compared with exvivo gene therapy, IO infusion could efficiently in situ transduce HSCsto avoid the lengthy and complicated procedures of cell manipulation andtransplantation. Significantly, the herein described approach avoids therequirement for use of fully or partially myeloablative conditioningregimens required for successful ex vivo gene therapy.

Compared to other in vivo LV-mediated gene therapy approaches, IOdelivery is unique. Slow injection (e.g., at a rate of 2 μL/min to 13μL/, or at 10 μL/) of LVs into bone marrow cavity can minimize or evenavoid the systemic toxicity caused by intravenous delivery of LV, AAVand other candidate gene therapy vectors. In addition, compared withhepatocyte-targeted LV gene transfer which induced high risk of antibodyformation after FVIII is secreted from hepatocytes into circulation, theplatelet-targeted FVIII gene therapy generated FVIII stored in plateletsand protected from processing by antigen presenting cells andneutralization by anti-FVIII inhibitors. During bleeding, FVIII wasreleased by activated platelets on-site to efficiently enhance clotformation. Bleeding phenotype is partially corrected in HemA mice withor without pre-existing anti-FVIII inhibitors. Additionally, targetingfactor VIII expression to platelets for HemA gene therapy did notincrease thrombotic risks but promoted immune tolerance in HemA mice.Therefore, the described strategy of IO LV delivery targeting FVIIIexpression in platelets is highly promising to treat HemA patients withor without inhibitors.

However, in vivo LV gene therapy brought associated immune responsesthat may decrease LV transduction efficiency and eliminate LV transducedcells. In immune competent mice given IO infusion of MND-GFP-LV, thepercentage of GFP⁺HSCs decreased over time (FIG. 1B). When the immunecompetent mice were given Dex before and after IO delivery of LVs, theaverage percentage of GFP⁺BM cells was 3-fold of that in control mice(FIG. B), which agreed with the finding that Dex treatment couldeffectively suppress inflammatory cytokine production after intravenousinfusion of LVs (Agudo et al., Mol. Ther. 20:2257-2267, 2012). Transientdepletion of CD8⁺ T cells by anti-CD8α mAbs in immune competent mice(FIGS. 8A, 8B) also produced higher percentage of GFP⁺HSCs over 7 weeksafter LVs infusion compared with control mice (FIG. 2C). Moreimportantly, combined drug treatment with Dex and anti-CD8α mAbs in theimmune competent mice achieved up to 14% GFP⁺HSCs in bone marrow morethan 5 months after viral infusion (FIG. 2D). Furthermore, in HemA micegiven IO infusion of G-F8/N6-LV, combined drug treatment improvedhemophilia phenotype correction, especially evaluated by blood flow rateassay (FIG. 3B). These agents in combination synergistically augmentedLV transduction. However, gradual decrease of GFP⁺HSCs was stillobserved in combined drug treated mice over a long period of time,suggesting that other immunomodulation regimens can potentially furtherenhance the persistence of transgene expression following IO delivery ofLVs.

A modified FVIII cDNA producing FVIII protein with higher expressionlevel, enhanced bioactivity and longer half-life would be desirable forsuccessful gene therapy of hemophilia A. Compared with human FVIII,canine FVIII is functionally more active and porcine FVIII expresses athigher levels. Novel recombinant FVIII cDNAs are thus designed withmutations at specific sits replacing with porcine- orcanine-corresponding amino acids to increase secretion and functionalityon the basic frame of human VIII sequence. Compared with F8/N6, F8X10K12directed by a ubiquitous promoter produced a 25-fold increase of FVIIIactivity in HemA mice following hydrodynamic injection (FIG. 4B) andexpressed higher levels of FVIII in LV-transduced 293T cells (FIG. 4C).Compared with mice treated with IOdelivery of G-F8/N6-LV directed by aplatelet-specific promoter, G-F8X10K12-LV treated mice produced higherFVIII expression levels in platelets (FIG. 5B). However, anti-FVIIIantibodies were induced likely due to higher levels of FVIII secretioninto the circulation (FIG. 5E).

It has been reported by that high levels of FVIII expression inplatelets could induce platelet apoptosis. Thus, the describedplatelet-targeted gene therapy of hemophilia A focused further onenhancing bioactivity rather than expression levels. The cDNA of a FVIIIvariant, F8/N6K12RH with higher activity contributed by a R1645Hmutation (Siner et al., Blood 121:4396-4403, 2013) and K12 mutations inlight chain, was incorporated into the LV construct. Compared withF8/N6, F8/N6K12RH directed by a ubiquitous promoter produced a 2.7-foldincrease of FVIII activity in HemA mice (FIG. 6A). Importantly,hemophilia phenotype correction in G-F8/N6K12RH-LV plus combined drugtreated mice was significantly improved compared with G-F8/N6-LV treatedmice, without the generation of anti-FVIII inhibitors (FIG. 6B and 6C)

FVIII ectopic expression in platelets provides an effective pathway forgene therapy of HemA patients, especially for patients with pre-existinganti-FVIII inhibitors as a result of frequent intravenous infusion ofFVIII. FVIII stored in platelets is not only protected from beingneutralized by inhibitors in peripheral blood, but also significantlyimproves the bleeding phenotype even with as low as 1%-5% of peripheralblood platelets containing FVIII. The therapeutic effects ofplatelet-FVIII was characterized by several FVIII function assaysincluding in vitro assays such as chromogenic assay (Shi et al., Mol GenMetabol. 79:25-33, 2003; Baumgartner et al., J Thromb & Haemostas.13:2210-2219, 2015) and thrombin generation assay (Monroe et al.,Arterios Thromb Vas Biol. 22:1381-1389, 2002), and in vivo assays suchas tail clipping assay (Wang et al., Mol Therapy 23:617-626, 2015; Shiet al., J Clin Invest 116:1974-1982, 2006) and FeCl₃ carotid arterythrombosis assay (Yarovoi et al., Blood 105:4674-4676, 2005; Yarovoi etal., Blood 102:4006-4013, 2003). FVIII-containing platelets wereisolated for use in in vitro assays. Isolated FVIII-containing plateletswere lysed, and released FVIII functional activity was detected by achromogenic assay using a Coatest FVIII:C/4 kit. However, results fromthe chromogenic assay could not accurately predict therapeutic efficacybecause of the very low level of platelet-FVIII even in transgenic mice(the highest platelet-FVIII level of 11 mU/10⁸ platelets in LV18^(tg+/+)mice, described in Baumgartner et al., J Thromb & Haemostas.13:2210-2219, 2015). In place of the chromogenic assay, the thrombingeneration assay was used where FVIII-containing platelets were directlyadded into HemA PRP and function was expressed as three parameters (lagphase time, peak height thrombin and total generated thrombin). LVtreated mice had shorter lag phase time compared to HemA controls,suggesting that FVIII-containing platelets played an important role inthe early stage of clot formation by accelerating thrombin generation.The tail clip assay of treated mice revealed IO infusion of LVssignificantly corrected bleeding phenotype compared to HemA controlmice. However, therapeutic effects between a combined drug regimen+LVstreatment and LVs only treatment could not be distinguished with thisassay. Therefore, the FeCl₃ carotid artery thrombosis assay, where bloodflow rate was monitored after local damage to the carotid artery, wasused. Compared with the modified tail clipping assay, FeCl₃ inducedcarotid thrombosis appears to be more sensitive to monitor the role ofplatelet-FVIII in enhancing local blood clot formation in arteries, andcapable of distinguishing therapeutic differences in the two treatmentgroups. Results from this assay showed that platelet-FVIII was effectivein arterial clotting and that a combination drug regimen with new FVIIIvariant, F8/N6RHK12, achieved phenotypic correction of HemA in mice.

In conclusion, persistent FVIII gene expression in platelets wasachieved by IO infusion of G-F8/N6K12RH-LV into HemA mice pretreatedwith a combination of Dexamethasone and anti-CD8α monoclonal antibody.Transient suppression of immune responses to LVs and LV-transduced cellsincreased LV transduction efficiency and produced long-term stabletransgene expression. F8/N6K12RH with an enhanced bioactivityeffectively improved the therapeutic effect of FVIII containingplatelets in phenotype correction assay. The function of platelet-FVIIIisolated from LV-treated mice was confirmed using a thrombin generationassay. Phenotypic correction of hemophilia was demonstrated using bloodclotting assays with tail clip and carotid artery injury models. Thisapproach may provide a readily translatable treatment for hemophilia inlarge animals and clinical trials.

Example 2. Intraosseous Delivery of Lentiviral Virus Acts as aTherapeutic Strategy for In Vivo Gene Therapy of Hemophilia

As discussed above, lentiviral vectors (LVs) delivered by intraosseous(IO) infusion at a precisely controlled speed can efficiently transducebone marrow hematopoietic stem cells (HSCs) in mice. IO infusion of LVcarrying a human FVIII/N6 transgene driven by a platelet-specific Gp1bαpromoter into hemophilia A (HemA) mice produced FVIII stored in plateletα-granules. These platelet FVIII partially corrected the bleedingphenotype over five months in HemA mice with or without pre-existinganti-FVIII inhibitors.

In the current example, IO delivery of LVs was applied in humanized NSGmice to establish a translational research model for in vivo genetherapy of hemophilia. Firstly, whether high level transgene expressioncould be achieved in human megakaryocytes (Megs) was examined. HumanCD34⁺ cells were transduced with Cocal-MND-GFP-LV (M-GFP-LV, MOI=25) orCocal-Gp1bα-GFP-LV (G-GFP-LV, MOI=25) and then cultured withMegakaryocyte Expansion Supplement (MES). 14 days after LV transduction,high level of GFP expression (36%) was detected in both M-GFP-LVtransduced Megs (CD41⁺ cells) and non-Megs (CD41⁻ cells). Furthermore,in G-GFP-LV-transduced human cells, GFP specifically expressed in Megs,but not in other types of cells derived from HSCs.

Next, FVIII specific expression in human Megs was evaluated inCocal-GP1bα-F8/N6-LV (G-F8/N6-LV) transduced human CD34⁺ cells. FVIIIexpression level in G-F8/N6-LV transduced human cells increased from 7%to 28% when the LV doses increased from MOI=10 to 50 on day 21 after LVtransduction. These results indicated that platelet-specific promoter,Gp1bα, could specifically guide high level transgene expression in humanMegs.

Transgene expression in humanized NSG mice following IO delivery of LVswas also investigated. Humanized NSG mice were created by I.V. infusionof 1×10⁶ human CD34⁺ cells into 6-week female NSG mice. After 8 weeks,G-GFP-LV, G-F8/N6-LV and Cocal-GP1bα-F8X10-LV (G-F8X10-LV) weredelivered into the humanized mice by IO infusion and transgene (GFP orhuman FVIII) expression was evaluated in human platelets. However, humanplatelets in blood of humanized mice rapidly decreased to undetectablelevels following human cells engraftment, possibly due to recognitionand destruction by murine macrophage cells.

In order to examine transgene expression in human platelets, the micewere given a series of clodronate liposome treatments (CLT) (0.7mg/animal, Day 0, 2, 7, 13), which was reported to transiently depletemurine macrophage cells. CLT treatment induced recovery of humanplatelets from an undetectable level to the level comparable to thepercentage of engrafted human cells in total PBMCs. In addition, similarlevel of GFP expression was detected in both human and murine plateletsof CLT+G-GFP-LV treated mice. Most importantly, higher platelet FVIIIlevel was found in CLT+G-F8/N6-LV or G-F8X10-LV treated mice thanG-F8/N6-LV or G-F8X10-LV only treated mice. Over 10% of the human cellsexpressed FVIII when human CD34⁺ cells were isolated from theG-F8/N6-LV-treated humanized mice and cultured with MES for 6 days.Next, LV integration sites in isolated human cells were examined byLAM-PCR.

Based on the described studies, IO infusion of LVs can be a valuablestrategy for in vivo gene therapy of hemophilia A and other bloodclotting disorders that would benefit from the expression of FVIII.

Example 3. Intraosseous Delivery of Lentiviral Virus Produces HumanPlatelet-Specific Factor VIII in Humanized NSG Mice

This example establishes proof-of-principle research models in humanizedNSG mice for translational applications of this novel strategy in humanclinics.

Hemophilia A (HemA) with a deficiency of functional plasma factor VIII(FVIII) is an ideal disease candidate for gene therapy to attainlong-term therapeutic FVIII levels.

Described herein is a new clinically translatable strategy for treatingHemA. In this strategy, self-inactivating lentiviral vector (LV)carrying FVIII transgene driven by a platelet-specific promoter Gp1bα(G) (G-FVIII-LV) was delivered into HemA mice without preconditioningvia intraosseous (IO) administration. G-FVIII-LV can effectivelytransduce hematopoietic stem cells (HSCs). Then, FVIII speciallyexpressed and stored in platelet α-granules can partially correctbleeding phenotype over five months in immune-competent HemA mice withor without pre-existing anti-FVIII inhibitors (Wang et al., Mol. Ther.,23(4):617-626, 2015).

Human CD34+ cells were cultured in SFEM II supplemented with cytokinecocktail for expansion of human hematopoietic cells (CC110), orserum-free culture supplement for expansion of human megakaryocytes(Meg) for 7 and 13 days (FIG. 12A). Human CD34+ cells cultured in SFEMII+Meg were transduced with M-GFP-LV or G-GFP-LV (FIG. 12B).Megakaryocytes and GFP expression on day 7 were detected by flowcytometry.

6-week NSG mice were retro-orbitally injected with 1×106 human CD34+cells one day after preconditioning with busulphan (25 mg/kg). After 13weeks, bone marrow (FIG. 13A), spleen (FIG. 13B), and blood (FIG. 13C)were collected. Human CD34+ cells in bone marrow, and human CD45+ cellsand murine CD45+ cells in bone marrow, spleen and blood were detected byflow cytometry.

M-GFP-LV was intraosseously infused into humanized NSG mice, which weregenerated by retro-orbital injection of human CD34+ cells 9 weeks ago.Four weeks later, the mice were sacrificed and bone marrow and spleenwere isolated. GFP expression in total cells, human CD45+ and murineCD45+ cells in bone marrow (FIG. 14A) and spleen (FIG. 14B) was detectedby flow cytometry.

Conclusions

G-CSF mobilized human CD34+ PBSCs cultured with SFEM II+Meg successfullydifferentiated into megakaryocytes in vitro, and transgene expressed inhuman cells after LV transduction.

Humanized mice were generated by retro-orbital injection of G-CSFmobilized human CD34+ PBSCs into preconditioned NSG mice. In the longterm, human CD34+ cells successfully renewed and differentiated intohematopoietic lineage cells in vivo.

GFP expressed in human cells of humanized NSG mice after intraosseousinfusion of LVs. Next, humanized NSG mice will be treated withintraosseous delivery of FVIII-LVs.

The IO delivery of LVs technology was applied in humanized NSG mice toestablish a translational research model for in vivo gene therapy ofhemophilia. Efficient transduction of human CD34+ cells to producemegakaryocyte-specific FVIII expression by G-F8-LV was demonstrated. Inaddition, it was demonstrated that following IO delivery of G-F8/N6-LVinto humanized NSG mice, human CD34⁺ cells isolated from the treatedmice showed significant FVIII expression. LV integration sites inisolated human cells can be examined by LAM-PCR, confirming the safetyof this strategy. These results further confirm that IO infusion of LVscould be a valuable strategy for in vivo gene therapy of hemophilia A.

Example 4: Evaluation of the Efficacy and Safety of IntraosseousDelivery of Platelet-Specific Factor VIII-Lentiviral Vectors as an InVivo Gene Therapy for Hemophilia in Humanized NSG Mice

Hemophilia A is a genetic disorder resulting in a deficiency infunctional factor VIII (FVIII) secreted in plasma and presents as anideal disease model for gene therapy. As described above, an in vivointraosseous injection (IO) therapy has successfully developed todeliver FVIII/N6 lentivirus (G-F8VIII-LV) driven by theplatelet-specific Gp1bα promoter. Viral transduction of hematopoieticstem cells (HSCs) ensures long-term continued maintenance of the genetherapy. FVIII is then produced and stored in the α-granules ofplatelets, avoiding detection by the immune system, making this therapyaccessible to patients who have already developed inhibitory antibodiesto FVIII. IO delivery of G-F8-LV partially corrected the hemophiliaphenotype in mice for at least 5 months. The aim of the current studywas to assess the safety and efficacy of IO delivery of G-FVIII-LV inhumanized mice to assure the feasibility of translating this therapy toclinical models.

In vitro studies were first conducted to confirm successful transductionand appropriate expression of G-F8-LV and G-GFP-LV in human CD34+ cellsisolated from G-CSF mobilized donors. Cells were cultured in StemSpan™Serum-Free Expansion Medium (SFEM) supplemented either with cytokinecocktail (CC110) to expand hematopoietic stem cells or withMegakaryocyte expansion supplement (MEG) following transduction topromote differentiation into megakaryocytes. After 7 days of growth,cells cultured in MEG had a higher percentage of both CD41a+ andlate-stage differentiated CD41a+CD42b+ megakaryocytes than thosecultured in CC110, indicating preferential differentiation intomegakaryocytes in this media. Higher FVIII and GFP expression was alsoobserved in MEG cultured cells, suggesting successfulmegakaryocyte-specific expression, as directed by the Gp1bα promoter.

These LVs were then tested in vivo in humanized NSG mice. NSG mice withand without the W-41 mutation in the c-Kit gene were preconditioned withbusulfan (25 mg/kg) 24 hours before the transplantation of hCD34+ cellsvia retro-orbital (RO) injection. Eight weeks later, higher engraftmentof hCD34+cells was observed in the W-41 NSG cohort compared with NSGmice, averaging greater than 80% engraftment in bone marrow. Humanplatelet count was also higher in the W-41 NSG mice.

IO delivery of LVs was next performed in cohorts of transplanted mice.Clodronate liposome treatments increased human platelet counts, allowingfor detection of notable FVIII expression in platelets isolated fromPBMCs of mice treated with G-FVIII-LV. Bone marrow was isolated severalweeks after the IO injection, and engrafted human HSCs were isolatedusing the AutoMACS CD34 MicroBead Kit. The isolated cells weresubsequently cultured in MEG and expressed significant levels of FVIIIand GFP after 14 days.

The genomic DNA (gDNA) of hCD34+ cells transduced by G-GFP-LV andG-F8-LV respectively was isolated at various MOls and the bone marrow oftwo groups of mice that had undergone IO injections with these two viralvectors respectively, and marking number was assessed by qPCR.

Using LAM-PCR, integration sites of the lentivirus are being analyzed toassess safety of this therapy. It is expected that there will be noclonal expansion. The integration sites are predicted to be random andpolyclonal.

Given its efficacy in humanized mouse models, IO delivery ofplatelet-specific FVIII presents as a promising in vivo gene therapy forcorrecting the hemophilia phenotype.

Example 5. Correction of Canine Hemophilia A Via Intraosseous Deliveryof a Platelet-Specific Factor VIII-Lentiviral Vector

This example describes development of a preclinical protocol oftreatment of HemA dogs using interosseous (IO) delivery of lentiviralvectors (LVs).

Background: Hemophilia A (HemA) results from a factor VIII (FVIII)deficiency. Traditional protein infusion to treat HemA is costly,inconvenient, and short-term. Intraosseous (IO) gene therapy viadelivery of lentiviral vectors (LVs) into bone marrow targeting FVIIIexpression in platelets successfully treated HemA mice with or withoutinhibitors. To facilitate the translation of this novel strategy toclinical application, treatment of HemA dogs via IOgene therapy wasinvestigated.

Methods: The VSVG-pseudotyped G-cFVIII-LV incorporating aplatelet-specific promoter Gp1bα and canine FVIII gene at either2.51*10⁸ ifu/kg or 1.34*10⁹ ifu/kg was injected into tibia bones of HemAdogs M80 and R04 separately. The injection volume of LV was 4.5 ml foreach dog. Gene expression of cFVIII was examined in platelets and plasmaisolated from LV-treated dogs by ELISA and aPTT assays. The correctionof HemA phenotype was evaluated by whole blood clotting time (WBCT) andthromboelastography (TEG) testing.

Results: Canine FVIII can be detected in platelets with the highestexpression at 30 mU/108 platelets (FIG. 15). No FVIII expression wasdetected in the dog plasma. WBCTs were shortened in multiple time pointsshortly after IO gene therapy (FIG. 16A). Similarly, all four parameters(R time, K time, α angle and MA) examined by TEG indicated partialcorrection of blood clotting (FIG. 16B). Furthermore, the IO genetherapy was well tolerated and didn't produce any toxicity in treateddogs as evaluated by CBC and blood chemistry analysis.

Conclusion: This example establishes a representative IO gene therapyprotocol to treat HemA mammals (exemplified by dogs) successfully. Thisstudy provides a strategy for safe and effective application of genetherapy in vivo for treating HemA patients.

Example 6: Intravenous Delivery of Lentiviral Vectors to TreatHemophilia A in a HSCs Mobilization Mouse Model

This Example describes development of a novel in vivo approach togenetically modify hematopoietic stem cells (HSCs) in peripheral bloodby lentiviral vectors (LVs) for the treatment of Hemophilia A.

Primitive HSCs entering peripheral blood from bone marrow (BM) werefirst mobilized by administering a combination of G-CSF and AMD3100 inmice, which was followed by intravenous injection (IV) of lentiviralvectors carrying FVIII gene (LVs). Gene delivery efficiency was theninvestigated by using four types of Lentivirus Vehicles (LVs) encodingthe GFP or FVIII gene, either driven by ubiquitous promoters or by amegakaryocyte specific glycoprotein-1 bα (Gp1bα) promoter (MND-GFP-LV,G-GFP-LV, EF-FVIII-LV, G-FVIII-LV). The results show that a single IVinjection of LVs could significantly increase transduced HSCs in bonemarrow of HSCs mobilized mice. Meaningfully, IV delivery of G-FVIII-LVsachieved long-term FVIII expression in platelets and corrected thehemophilia A phenotype partially without signs of inhibition of FVIIIexpression. Therefore, the in vivo lentivirus delivery strategy has highpotentials for an effective treatment of FVIII deficiency in patients.

Introduction: Hemophilia A with deficiency of FVIII is a rare disease asdefined by World Health Organization. Hemophilia A (HemA) is a seriousbleeding disorder caused by deficiency in Factor VIII gene. Individualswith hemophilia A suffer from repeated bleeding episodes, which cancause chronic joint disease. After expensive treatment with therecombinant active FVIII, 30% of individuals develop an immunogenicresponse in the form of neutralizing antibodies against FVIII (Miao,Thrombosis Journal 14:41, 2016).

Gene therapy is a good strategy to treat HemA (Roth et al., New EnglandJournal Med 344:1735-1742, 2001; White, Thrombosis and Haemostasis86:172-177, 2001; Naldini, Nature 526:351-360, 2015; Yarovoi et al.,Blood 102:4006-4013, 2003). Recently, clinical trials by biotechnologycompanies show very promising results. AAV vectors are used to deliveryFVIII, hybrid liver-specific promoter (HLP) specifically driving itsexpression in liver for restoring FVIII in plasma (Pasi et al.,Haemophilia 22:151-152, 2016). However, there is a risk of triggeringimmune responses to generate anti-FVIII inhibitory antibody, whicheventually results in reducing/eliminating of the FVIII in plasma (Ertlet al., Human Gene Therapy 28:328-337, 2017; Doering et al., MolecularTherapy: J Amer Soc Gene Therapy 15:1093-1099, 2007). In addition, quitemany patients have developed inhibitors against AAV vectors, thus thatsuch gene therapy method will not work.

Specific expression of FVIII in platelets is a decent gene therapymethod for the treatment of HemA. Wang et al delivered lentivirusconstruct containing the Gp1bα-FVIII via intraosseous infusion(Molecular Therapy: J Amer Soc Gene Therapy 23:617-626, 2015). Bloodclotting can be partially corrected by the treatment with plateletsspecific expression of the FVIII. There are also other investigators whoemploy Gp2bα promoter to drive FVIII expressions. Both of the methodsresulted in FVIII storing in platelets. However, the mice with LVsintraosseous delivery or transplantation of transuded HSCs didn't showFVIII inhibitors.

Using G-CSF and AMD3100 to mobilize primitive progenitor cells is anefficiency method of enforcing the HSCs in bone marrow into peripheralblood. Many studies confirmed that mobilized HSCs can be transduced byan integrating adenovirus vectors in vivo. Additionally, somepreclinical studies use the mobilization to enhance gene therapyefficacy. For example, others employed immune reconstitution inmobilized dogs with X-linked severe combined immunodeficiency disease inmobilization dogs. Combination mobilization and IV delivery lentivirusvectors is a good strategy. Therefore, the hypothesis of this study isthat mobilized HSCs from bone marrow to peripheral blood could betransduced by lentivirus carrying GFP or FVIII gene. Furthermore,platelets-specific promotor driving FVIII expression in platelets couldavoid the development of FVIII inhibitors.

Materials and Methods

Animals and Cells: All in vivo studies and HemA breeding have beenapproved by the Institutional Animal Care and Use Committee (IACUC) ofSeattle Children's Hospital, under IACUC Protocol No. 130001330. Allanimal procedures and animal care were performed according toinstitutional animal research guidelines and were also in compliancewith Seattle Children's Hospital Animal Management Regulations. HEK293Tcells were obtained from the ATCC. Cells were cultured in relevant cellculture medium supplemented with 10% (vol/vol) FBS, 1% (vol/vol) P/S, 1%(vol/vol) Glutamine and 1% (vol/vol) HEPES. Testing for potentialmycoplasma contamination was done by ELISA.

Mice mobilization and in vivo transduction: Male black/6 mice or maleHemA mice (6-8 weeks) are subjected to mobilization procedure. C-GSF wassubcutaneous injected 50 ng/mouse for 4 days. At the fifth day, AMD3100was injected in mice (5 mg/kg) via SC. Lentivirus infused at the fifthday after AMD3100 injection 30 minutes. Dexamethasone was dosed to miceintraperitoneally 16 hours and 2 hours before lentivirus injection. SeeFIG. 22B and 22C for additional details.

Lentivirus production: Lentiviral vectors (LVs) were produced asdescribed previously (Wang et al., Mol Ther 23(4)617-626, 2015); briefly12 μg Transgenes (pRRL-SIN-MND-eGFP-WPRE, pRRL-SIN-Gp1bα-eGFP-WPRE,pRRL-SIN-EF-1α-hFVIII/N6-WPRE, and pRRL-SIN-Gp1bα-hFVIII/N6-WPRE), 6 μgviral package plasmid (psPAX2), 3 μg of viral envelop plasmid (pMD2·G)were transfected HEK293T cells with 33 μg of polyethyleneimine (linear,MW ˜40,000, Polysciences, Inc, Warrington, Pa.). LV-containingsupernatants were concentrated ˜2000-fold by low-speed (8000 g)centrifugation for 16-18 hr at 4° C. Infectious vector stocks werestored at −80° C. Viral titers were determined by real-time quantitativePCR (qPCR) as described previously (Kerns et al., Blood 115:2146-2155,2010). Briefly, 2×10⁵ Nalm-6 pre-B cells were transduced with serialdilutions of concentrated virus. Cells were harvested 7 days later,washed twice with PBS and used for genomic DNA (gDNA) extraction. Viraltiters (ifu/ml) were calculated from the transduction rate determined bygDNA qPCR. Average titers were 2.2×10⁹ ifu/ml, 2.5×10⁹ ifu/ml, 2.5×10⁹ifu/ml, and 1.1×10⁹ ifu/ml for M-GFP-LV, E-F8-LV, G-GFP-LV, and G-F8-LV,respectively.

Flow cytometers: HSCs or LSKs were stained and gated bylineage—ckit+scal+ in BM cells or PBMCs. Platelets were isolated fromPBMCs by centrifuging method. The platelets were separated by isolationbuffer Fico/Lite™-Platelets (Atlanta Biologicals) after 90 g/min, 8 minspinning. The isolated platelets were stained by CD42d. B, T, andmyeloid cells were stained by surface maker B220, CD3, and CD11b(eBioscience San Diego, Calif.). For FVIII expression in platelets,after surface staining, platelets were fixed by fix and perm buffer(BD). Mouse anti-hFVIII monoclonal antibody (ESH8, American Diagnostica,Seabrook, Tex.) was used for primary antibody, followed by secondantibody secondary goat anti-mouse Ig FITC (BDBiosciences). Flowcytometric analysis was conducted using LSRII (BD Bioscience) and thedata were analyzed using FlowJo (Ashland, Oreg.).

FVIII activities and FVIII antibody assays in Plasma: hFVIII activitiesin mouse plasma were analyzed using aPTT, and anti-hFVIII antibodieswere measured by hFVIII Bethesda inhibitor assay as previously described(Miao et al., Blood 114:4034-4044, 2009; Kasper et al., Thromb DiathHaemorrh 34:612, 1975).

Clotting functional assay: Rotation thromboelastometry (ROTEM®)functional clotting assay was employed to characterize the bloodclotting function, using a computerized ROTEM® four-channel device(Instrumentation Laboratory, USA). Mice blood was collected via chinbleeding and thoroughly mixed with 10% sodium citrate. ROTEM® assayswere performed under Intern program by the mini cup protocol. The bloodcollected from control and gene therapy groups were subjected to run forone hour to investigate coagulation time (CT), clot formation time(CFT), α-angle, and max clot firmness (MCF) on ROTEM®.

Copy number detection: Genomic DNA was isolated in BM cells by QiagenDNA mine Kit. One copy Nalm-6 cell gDNA was diluted as a standard. GAGprimers and Probe were synthesized by Invitrogen. Forward Sequence:GGAGCTAGAACGATTCGCAGTTA (SEQ ID NO: 5); Reverse Sequence:GGTTGTAGCTGTCCCAGTATTTGTC (SEQ ID NO: 6). Probe:ACAGCCTTCTGATGTTTCTAACAGGCCAGG (SEQ ID NO: 7)

Quantitation of hFVIII mRNA in mouse liver, spleen and lung wasperformed by qRT-PCR using Power SYBR Green PCR Master Mix (Appliedbiosystem, Warrington, UK) according to the manufacturer's instructions.Firstly, total RNA was extracted from hydrodynamically (HD) injected(pBS-HCRHPI-FVIIIA48) mouse livers, and IO treated (G-F8-LV) or naivemouse livers, spleens and lungs using TRIzol® Reagent (Lifetechnologies, Grand Island, N.Y.). Then cDNA was generated using cDNAsynthesis kit (Invitrogen, Carlsbad, Calif.). hFVIII specific primers(Forward Sequence: CCAGAGTTCCAAGCCTCCAACA (SEQ ID NO: 8); ReverseSequence: GGAAGTCAGTCTGTGCTCCAATG (SEQ ID NO: 9)) for evaluating theFVIII transcripts and Rpl19 primers (Forward Sequence:GCATCCTCATGGAGCACAT (SEQ ID NO: 10); Reverse Sequence:CTGGTCAGCCAGGAGCTT (SEQ ID NO: 11)) for assessing the controlhousekeeping gene transcripts were used in qRT-PCR experiments. ThehFVIII mRNA expression in HD mouse livers was used as the positivecontrol (RQ=1) to calibrate the hFVIII mRNA expression levels in IOtreated or naive mouse livers, spleens and lungs.

Results Increases of GFP-Marked Hematopoietic Lineages in MobilizationMice after LV Transduction

LV containing the ubiquitous promoter MND to drive GFP expression inmobilization mice model was used to evaluate the transduction efficiencyvia IV delivery of lentivirus. The mobilization drugs (G-CSF andAMD3100; see FIG. 22C) were utilized to mobilize HSCs from BM toperipheral blood. The mobilization resulted in more than 100 foldsincrease of LSK cells entering blood (FIG. 17A). Then, IV deliveryMND-GFP-LV was initiated by retro orbital injection. The highest GFPexpression in the whole PBMCs is at Day 7 in the mobilization groupafter administration of MND-GFP-LVs (FIG. 17B). GFP-positive LSK cellsin blood can be detected even in the 10th week (FIG. 17C). MND-GFP-LVsalso can transduce myeloid cells, T cells and B cells, but a fasterdecline of GPF was observed in these cell types in the mobilizationgroup (FIGS. 17D-17F).

The transduction status in BM was also analyzed after MND-GFP-LVsinjection in Mobilization group and non-mobilization group. The GFP MFI(mean fluorescence intensity) of total mononuclear cells in BM displaysa significant difference between mobilization group and non-mobilizationgroup 10 weeks post LVs delivery (FIG. 17G).

GFP expression in HSCs of BM can be detected in both groups by flowcytometer after 10 weeks. MFI of the GFP+ signal in the mobilizationgroup is 5 times higher than that in the non-mobilization group (FIGS.17H). These results confirmed that IV delivery of LVs can efficientlytransduce HSCs mobilized in the peripheral blood, and that thetransduced HSCs home back to BM from the circulation.

Gp1bα Promoter Drives Sustained GFP Expression in Platelets AfterG-GFP-LV Injection in Mobilized Mice Model

As in this case of LVs transducing HSCs successfully by systemicdelivery under the condition of mobilization. Gp1bα promoter would driveGFP expression in megakaryocytes and storage Platelets. To evaluate thepossibility, G-GFP-LVs was injected in mobilization and non-mobilizationgroup via IV. The peak of GFP expression in platelets was at the 2ndweek after G-GFP-LVs injection. Sustained GFP expression in plateletswas observed in about 0.2% platelets in mobilization group at 10 weeks(FIG. 18A). Meanwhile, GFP expression was not detectable innon-mobilization group after LVs delivery. G-GFP cannot be detected inT, B and Myeloid Cells, which demonstrated that Gp1bα promoter expressedonly in megakaryocytes. These results indicated that transductionefficiency of G-GFP-LVs is significantly enhanced in HSCs aftermobilization to allow increased GFP expression in platelets. FIG. 18B isthe representative flow data; FIG. 12A is the summary of the results.

Ubiquitous expression of E-F8-LV vectors via IV injection induceformation of FVIII antibodies in vivo

To demonstrate the therapeutic benefits of this novel strategy of LVsinfusion, we treated HemA mice by IV injection of the EF-hFVIII-LVs. Inboth mobilization group and non-mobilization group, the circulatoryFVIII activity in plasma was detected by APTT assay after 2 weeksE-FVIII-LVs treatment (FIG. 19A). We observed a decrease of FVIIIexpression to undetectable levels 6 weeks after LVs infusion, whichcorrelated with the appearance of anti-FVIII inhibitory antibodies (FIG.19B). Thus, it appears that FVIII expression driven by ubiquitouspromoters tends to induce formation of FVIII antibodies.

FVIII protein can be stored in platelets after G-FVIII-LV transductionin vivo

The approach of G-FVIII-LVs treating HemA emerged since the G promotercan drive GFP expression and stocked in platelets. Delivery ofG-FVIII-LVs for the expression of FVIII in platelets specifically hasthe potential to escape immune response against FVIII. To test thispossibility, G-FVIII-LVs was delivered via IV; there were 1.5-4%platelets containing FVIII in the mobilization group for 3 monthsexperimental period (FIGS. 20A-20B). By ELISA assay detection, FVIIIexpression was about 1.5 mU/10⁸ platelets (FIG. 20C). The copy number inBM 0.3 copy/cell by q-PCR detection (FIG. 20D). No mouse in themobilization group injected with G-FVIII-LVs displayed FVIII inhibitorin plasma.

Targeted FVIII Expression in Platelets Partially Corrects HemophiliaPhenotype

To determine if expression of FVIII in platelets could functionallycompensate Hemophilia, the ROTEM® (rotational thermoelectrometry) assaywas employed; this is a clotting assay to evaluate the blood clottingfunction by detecting the clotting time (CT), clot formation time (CFT),max clotting firmness (MCF) and a angle. The mice transduced byG-FVIII-LVs were subjected to ROTEM® assay after 6 months gene therapy.ROTEM® results confirmed a faster clotting time, shorter clottingformation time, and bigger a angle in the mobilization group comparedwith those in HemA mice. There were significant differences betweenmobilization and non-mobilization groups on a angle and MCF parameters.In the non-mobilization group, the blood clotting function did not showsignificant improvements, when compared with that in HemA mice (FIGS.21A-21D).

Discussion

To obtain long-term FVIII expression after single treatment, severalgroups have explored different approaches of gene therapy techniques. Atleast four gene therapy methods are in clinical trials. BMN270 and Spark8011 are in clinical trial phase II/III; these are gene therapy-basedmethods using AAV vector to delivery FVIII, with HLP promoterspecifically driving expression in liver for restoring FVIII in plasma(Pasi et al., Haemophilia 22:151-152, 2016). After the treatment, FVIIIare expressed in liver and secreted into blood, where it may triggerimmune responses to generate anti-FVIII inhibitory antibody.Additionally, there is also a risk of the AVV being cleaned by naturalinhibitor in the human body (Ertl et al., Human Gene Therapy 28:328-337,2017; Doering et al., Molecular Therapy: J Amer Soc Gene Therapy15:1093-1099, 2007). Gene therapy ex vivo studies also have somedrawbacks, even a long-term FVIII expression and correction hemophiliaphenotype were observed after implantation of transduced HSCs back torecipient animal model. Thus there is an ongoing need for theherein-described intravenous delivery of lenti-vectors in HSCsmobilization mice model, which can avoid development of inhibitors, orHSCs culture in vitro.

LVs delivery via intravenous injection was performed before. In 2006,Carbonaro and his coworkers used LV vectors via intravenous injection inmouse neonatal ADA knockout mice, and detected copy number of integratedvectors in liver and lung (Carbonaro et al., Mol Ther 13(6):1110-1120,2006). In adult mice, IV injection was performed with detection of lowcopy number in BM. From those studies we learned that Lentivirusdelivery via IV rendered less efficiency transduction in the HSCs of BM.The reason is that Lentivectors were eliminated by macrophages orKupffer cells in the peripheral blood (Brown et al., Blood109(7)2797-2805, 2007), there were less chance that vectors wouldmigrate into bone marrow to transduce HSCs. Mobilization of HSCscombining with IVs injection is a good strategy, which promoteefficiency of HSCs transduction, as the location of transduction waschanged from BM to peripheral blood. Those HSCs transduced by LVscarrying FVIII would go back to BM and stay in the niche of BM forfurther regeneration and differentiation.

Indeed, this approach increased the efficiency of LV transductionsignificantly. Data herein showed that MFI is 5-fold higher inmobilization group than that in the non-mobilization group after LVinjection. However, when the ubiquitous promoter was used to drive theFVIII expression secreting in plasma, either mobilization ornon-mobilization group displayed higher titer of FVIII inhibitor.

FVIII driven by GP1b promoter is advantageous because it is expressed inthe megakaryocytes cells, and storage of the FVIII in platelets preventsit from exposure to immunity system. When a bleeding event occurs,platelets will be activated to release the FVIII and therefore join thecoagulation pathway.

To demonstrate that G-FVIII-LVs also can achieve long term therapy inHemA mice, the efficacy study was tracked up to 6 months in mousemodels. The blood clotting functional data demonstrated that HemAphenotype was corrected partially for a long period. Low titer of FVIIIinhibitor was observed in the non-mobilization group, although there wasno significate difference compared with that mobilization group. Invitro experiment demonstrated that GP1b promoter can work in myeloidcell.

This study explored a novel strategy for gene therapy of HemA viadelivery of FVIII-LVs specifically transduced HSCs by intravenousinjection. This is a novel approach and offers several advantages overprior strategies, including: The IV delivery is straight forward andconvenient. It does not need complicated ex vivo transplantation or cellculture in vitro. In addition, it achieves long-term efficacy bytargeting HSCs. FVIII are stored in platelets, there are less chance toinduce neutralization of FVIII by antibodies.

Example 7: A Novel Strategy of Lung-specific Gene Therapy for theTreatment of Hemophilia A via Intranasal Delivery of Lentiviral VectorsEncoding Factor VIII

Recently, emerging evidence showed that lung is a reservoir forhaematopoietic progenitors/stem cells (HSCs) in addition to bone marrow(Lefrancias et al., Nature 544(7648):105-109, 2017). The new studyreported that platelets can be produced from megakaryocytes located inthe extravascular space inside the lung. Furthermore, data in the aboveExamples demonstrated that external factor VIII (FVIII) gene driven bythe specific promoter GP1bα (G) could express FVIII protein inmegakaryocytes for long-term by transducing HSCs in mice. FVIII proteinstored in platelets can be released to facilitate clotting uponplatelets activation. Thus, intranasal (IN) delivery of lentiviralvectors (LVs) targeting FVIII gene expression in megakaryocytes in thelung have the potential to correct HemA mice phenotype.

G-GFP-LVs were used to test if IN delivery can produce GFP expression inmurine platelets. IN delivery of 24 μl G-GFP-LVs produced up to 0.075%positive GFP expression in platelets after 7 days. Although theefficiency was low, GFP expression could be detected by flow cytometer.There was a significant difference compared with the untreated controlgroup.

To improve the transduction efficiency, higher titers/volume of LVs wereused in mice pretreated with N-Acetyl Cysteine and Dexamethasone. AfterIN delivery of increased dosages of G-FVIII-LVs into HemA mice under theoptimized protocol: Mice were treated with 130 mg/kg N-acetyl cysteine(NAC) via intravenous injection for 2 days and 10 mg/kg Dexamethasonevia intraperitoneal injection 16 hrs before and 2 hrs after IN. Micewere administered with 40 μl G-FVIII-LVs (1×10⁹ IFU/ml)/mouse via IN,then mice slept for 5 mins under isoflurane. FVIII expression inplatelets was examined by intracellular staining and FVIII ELISA at day7 post treatment. There were 0.23% FVIII positive platelets incirculating platelets and FVIII expression can be detected up to 0.3mU/10⁸ platelets.

Functional clotting formation was also tested by ROTEM®. The clottingparameters obtained from IN gene therapy mice were comparable to thoseof wild type mice, including Clotting Time (CT), Clotting Formation Time(CFT), a angle, and Max of Clotting formation (MCF). Moreover,significant difference in the CT values was observed between HemA micegroup and IN gene therapy group (2641±1437(s) vs 587±354(s); p=0.032)after 7 days treatment. These data indicated successful transduction ofmegakaryocytes in the lung following IN delivery, leading to therapeuticcorrection of HemA.

Furthermore, FVIII positive platelets staining persisted at 0.23% fromday 7 to day 30 post treatment. The clotting parameters tested by ROTEM®after day 30 treatment were not significantly different compared withthe data after day 7 treatment, indicating potential transduction ofhematopoietic stems cells by LVs. In addition, formation of anti-FVIIIinhibitors was not detected by Bethesda assay.

These data showed that FVIII expressing platelets were successfullyproduced and were able to partially correct HemA phenotype in mice viaIN delivery of G-FVIII-LVs into the lung. A benefit of FVIII expressionand storage in platelet α-granules is to protect FVIII from naturalizingantibodies and to decrease the possibility of inducing inhibitoryantibodies. IN delivery of LVs encoding FVIII gene targeting lung is apromising option for therapeutic treatment of hemophilia patients.

Example 8: A Novel Strategy of Lung-specific Gene Therapy for theTreatment of Hemophilia A via Intranasal Delivery of Lentiviral VectorsEncoding Factor VIII

In this example, an embodiment of the herein described treatment methodis demonstrated, using intranasal delivery of LVs to treat hemophilia Amice .

Hemophilia A (HemA) disease is a deficiency of functional plasma factorVIII (FVIII). Recently, emerging evidence showed that lung is areservoir for haematopoietic progenitors/stem cells (HSCs) in additionto bone marrow. The new study reported that platelets can be producedfrom megakaryocytes located in the extravascular space inside the lung.

We utilized lentiviral vectors (LVs) driven by a Gp1bα promoter totransduce lung via intranasal delivery (IN) to generateplatelet-specific FVIII gene expression for the treatment of HemophiliaA.

The vectors used in this study are provided in FIG. 23. LVs weretransduced via intranasal delivery, then lung, bone marrow, andplatelets were harvested and analyzed for gene expression.

Results:

G-GFP expression in platelets by G-GFP-LVs transduction via intranasaldelivery is illustrated in FIG. 24A, 24B. Mice were administrated with24 μl G-FVIII-LVs (1×10⁹ IFU/ml)/mouse/day via IN for 3 days. (FIG. 24A)The flow cytometry schematic of GFP+ platelets. (FIG. 24B) GFPexpression in platelets was measured by flow cytometer after lentivirustreatment (N=4).

Expression of FVIII in platelets by G-FVIII-LV via intranasal deliveryis illustrated in FIG. 25A-25C. Mice were treated with 130 mg/kgN-acetyl cysteine (NAC) via intravenous injection for 2 days and 10mg/kg Dexamethasone via intraperitoneal injection 16 hrs before and 2hrs after IN. Mice were administered with 40 μl G-FVIII-LVs (1×10⁹IFU/ml)/mouse via IN, then mice slept for 5 mins under isoflurane. (FIG.25A-25B) FVIII expression was detected by intracellular staining andELISA assay in platelets after IN treatment (N=4). (FIG. 25C) Therepresentative schematic of the whole blood clotting by ROTEM in HemA,wide type, and LV-treated mice at 30 days after IN dosing.

Lung HSCs transduction by M-GFP-LVs is illustrated in FIG. 26. M-GFP-LVscan transduce hematopoietic stem cells in lung via intranasal delivery.

Conclusions: Transducing megakaryocytes in lung with lentiviral vectorsvia intranasal delivery achieved instant gene expression. NAC and Dextreatment helped LVs transduction efficiency. M-GFP-LVs can transducehaematopoietic stem cells in lung via intranasal delivery. Transducinghaematopoietic stem cells in lung by G-FVIII-LV via intranasal deliveryto achieve long-term partially cure of HemA.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient, orcomponent. As used herein, the transition term “comprise” or “comprises”means includes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient, or component not specified. The transition phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients, or components and to those thatdo not materially affect the embodiment. As used herein, a materialeffect would cause a statistically-significant reduction in transientexpression of a therapeutic protein within 7 days followingadministration of a disclosed nanocarrier to a subject.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the herein-citedreferences and printed publications are individually incorporated hereinby reference for their particular cited teachings.

It is to be understood that the embodiments disclosed herein areillustrative of the principles of the present invention. Othermodifications that may be employed are within the scope of theinvention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention may be utilized in accordancewith the teachings herein. Accordingly, the present invention is notlimited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the examples or when application of themeaning renders any construction meaningless or essentially meaningless.In cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

What is claimed is:
 1. A method of treating a subject in need of plasmaFactor VIII, comprising: administering to the subject a therapeuticallyeffective amount of an inflammation suppressor; administering to thesubject a therapeutically effective amount of a CD8+ T cell depletingagent; and administering to the subject a therapeutically effectiveamount of a composition comprising a lentiviral vector comprising anoptimized FVIII expression cassette expressibly linked to amegakaryocyte-specific promoter, wherein administration is via:intraosseous (IO) infusion; intravenous (IV) delivery; or intranasal(IN) or inhaled delivery.
 2. The method of claim 1, wherein theinflammation suppressor comprises dexamethasone (Dex).
 3. The method ofclaim 2, wherein the dexamethasone is administered in doses of 100 mg/kgat −24 h before, −4 h before, 4 h after, and 24 h after administrationof the LV.
 4. The method of claim 1, wherein the CD8+ T cell depletingagent comprises an anti-CD8 antibody.
 5. The method of claim 4, whereinthe CD8+ T cell depleting agent is an anti-CD8α mAb administered indoses of 4 mg/kg at −1 day before, 4 days after, and 11 days afterdelivery of the LV.
 6. The method of claim 1, wherein themegakaryocyte-specific promoter is a GP1b-alpha promoter.
 7. The methodof claim 1, which method does not comprise pre-conditioning ormyeloablative treatment of the subject.
 8. The method of claim 1,wherein the IO infusion is carried out at a rate of 2 μL/min to 15μL/min, 5 μL/min to 12 μL/min, or at no more than 10 μL/min.
 9. Themethod of claim 1, wherein the IO infusion is carried out at a rate of0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25mL/min, or at no more than 0.4 mL/min.
 10. The method of claim 1,wherein the IO infusion is carried out over a period of no more than 45minutes.
 11. The method of claim 1, wherein the optimized FVIIIexpression cassette comprises hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH(SEQ ID NO: 2).
 12. The method of claim 1, wherein the lentiviral vectorcomprises SEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant thereof.13. The method of any one of the previous claims, which is a method fortreating a hemostasis related disorder in the subject, and the subjectis in need of such treatment.
 14. The method of any one of the previousclaims, wherein the subject has hemophilia A (hemA), von Willebranddisease, bleeding associated with trauma or injury, thrombosis,thrombocytopenia, stroke, coagulopathy, disseminated vascularcoagulation (DIC), or over-anticoagulation treatment disorder.
 15. Themethod of any one of the previous claims, wherein the subject is a HemAsubject with preexisting anti-FVIII inhibitory antibodies.
 16. A methodof treating a subject in need of plasma Factor VIII, essentially asdescribed herein.